Microhomology-mediated end joining acts directly on replication forks to repair single-ended double strand breaks

Shibo Li; Yuqin Zhao; Youhang Li; Sameer Bikram Shah; Yanmeng Shi; Tran Nguyen; Zi Wang; Chia-Yu Chang; Anagh Ray; Te-Hsuan Bu; Salvatore Loguercio; Takayo Sasaki; Jonathan H Sussman; Hailong Wang; David M Gilbert; Mirit I Aladjem; Xiaohua Wu

doi:10.1016/j.molcel.2026.02.016

. Author manuscript; available in PMC: 2026 Jul 10.

Published in final edited form as: Mol Cell. 2026 Mar 16;86(7):1230–1246.e8. doi: 10.1016/j.molcel.2026.02.016

Microhomology-mediated end joining acts directly on replication forks to repair single-ended double strand breaks

Shibo Li ^1,², Yuqin Zhao ^2,^6,⁷, Youhang Li ^2,^6,⁷, Sameer Bikram Shah ^2,⁷, Yanmeng Shi ^2,⁷, Tran Nguyen ², Zi Wang ², Chia-Yu Chang ², Anagh Ray ³, Te-Hsuan Bu ², Salvatore Loguercio ⁴, Takayo Sasaki ⁵, Jonathan H Sussman ², Hailong Wang ⁶, David M Gilbert ⁵, Mirit I Aladjem ³, Xiaohua Wu ^2,^8,^*

PMCID: PMC13345754 NIHMSID: NIHMS2151773 PMID: 41844151

Summary

Replication stress, intrinsic to oncogenesis, often leads to fork breakage and double-strand break (DSB) formation. Conventionally, break-induced replication (BIR) is considered the primary mechanism for repairing replication-associated single-ended DSBs (seDSBs). Here, we demonstrate that microhomology-mediated end joining (MMEJ) acts directly to repair seDSBs at broken replication forks (fork-MMEJ), preferentially on the leading strands, and functions cooperatively with BIR. While promoted by Polθ, fork-MMEJ operates independently of MRE11/CtIP-mediated end resection, relies on RPA, and produces asymmetric deletion patterns, distinct from canonical MMEJ (cMMEJ) defined at replication-independent double-ended DSBs (deDSBs). ATR, activated as end resection proceeds, serves as a pivotal switch to suppress fork-MMEJ while promoting BIR. Combined inactivation of ATR and Polθ synergistically kills cancer cells under high replication stress with minimal toxicity to normal cells. Together, our study provides fundamental insights into the MMEJ mechanism and offers new strategies for cancer treatment.

eTOC blurb

Li et al. uncovered a new mechanism, fork-MMEJ, at broken forks to repair seDSBs, with characteristics distinct from the conventional MMEJ (cMMEJ). This study provides insights into replication-associated DSB repair, implicates microhomology-mediated chromosomal rearrangements in cancer, and offers potential therapeutic strategies to selectively target cancer cells.

Graphical Abstract

graphic file with name nihms-2151773-f0008.jpg

Introduction

Replication fork breakage can occur during S-phase due to replication stress (RS)¹ or when replication encounters single-strand breaks (nicks), which are among the most common endogenous lesions². Fork breakage often generates single-ended double strand breaks (seDSBs), with break-induced replication (BIR) serving as the primary repair mechanism^3,4. As RS is integral to oncogenesis^5–7, fork breakage-induced DSBs are a major source driving cancer-related genome instability. Microhomology sequences are frequently observed at cancer breakpoints^8–12, suggesting the potential involvement of microhomology-mediated end joining (MMEJ) in cancer-related chromosomal rearrangements^13–17; however, the underlying mechanisms are still elusive.

Canonical MMEJ (cMMEJ) is thought for repairing double-ended DSBs (deDSBs)^18–21, requiring short-range end resection by MRE11 and CtIP to expose microhomology (MH) sequences for annealing, followed by trimming of non-homologous (NH) tails, DNA synthesis from the 3’ end of MH, and end ligation (Figure S1A)^19,20. DNA polymerase theta (Polθ), encoded by the POLQ gene, plays a central role in cMMEJ^13–17. MMEJ is inherently mutagenic and often causes deletions and insertions (indels) accompanied by 1–6 bp of MH at the repair junctions^18–21.

Polθ deficiency sensitizes cells not only to ionizing radiation but also RS-inducing agents^14,22,23. Polθ binds to replication forks with binding increasing under RS^23–27. DSBs caused by RS can persist into mitosis and be repaired by Polθ-mediated MMEJ^28–31. Polθ is also involved in filling post-replicative single-stranded DNA (ssDNA) gaps and promoting microhomology-mediated gap skipping in BRCA-deficient cancer cells^24,25. In C. elegans, cMMEJ participates in post-replicational repair of broken forks ^22,32–35. Despite the key role of Polθ in the RS response, it remains unknown whether MMEJ can directly repair seDSBs upon fork breakage.

Fork breakage at nicks generates distinct DSB structures on leading versus lagging strands. Leading-strand breakage produces seDSBs through Cdc45/MCM/GINS (CMG) run-off, whereas lagging-strand breakage often converts seDSBs to deDSBs as CMG bypasses nicks^36,37, although seDSBs are also detected on broken lagging strands³⁸. Notably, nickase-induced seDSBs on lagging strands contain 3’ ssDNA overhangs, whereas those on leading strands are typically blunt-ended³⁸. These distinct DSB end structures raise the question of strand-specific repair. In yeast, acetylation is required only on the broken leading strands to facilitate HR³⁷. In mammalian cells, although HR repairs DSBs on both strands, it appears to be more strongly activated on broken lagging strands³⁹ and more dependent on BRCA1⁴⁰; yet the underlying mechanism driving this difference remains unknown.

Here, we discovered a new MMEJ activity that directly repairs fork breakage-induced seDSBs, which we term fork-MMEJ (Figure S1B), mechanistically distinct from cMMEJ at deDSBs. We also uncovered an elegant regulatory control mechanism by ATR that governs the use of fork-MMEJ and BIR.

Results

MMEJ is induced upon fork breakage after Cas9n cleavage.

We inserted our established EGFP-MMEJ reporter⁴¹ (Figure 1A top) into the AAVS1 locus in U2OS cells, flanked by bidirectional replication origins^42,43 (Figure S1C). To study MMEJ at broken forks, we used gRNA2 (g2)/Cas9^D10A to introduce a nick on the top strand of the reporter, 1 bp outside of the left MH (Figure 1A top). As replication approaches the nick from either side, fork breakage occurs on the leading strand, producing seDSBs (lead-seDSBs), or on the lagging strand, generating seDSBs that are subsequently converted to deDSBs (lag-seDSBs/deDSBs)^36,38 (Figure S1D). Thus, g2/Cas9^D10A-induced nicks are expected to generate a mixture of lead-seDSBs and lag-seDSBs/deDSBs in the reporter cell population.

We observed robust MMEJ activity at g2/Cas9^D10A-induced nicks in the EGFP-MMEJ reporter at the AAVS1 locus and other genomic sites, comparable to deDSB-induced MMEJ after g2/Cas9^WT cleavage (Figure 1A bottom and S1E top). This nick-induced MMEJ (g2/Cas9^D10A) was suppressed by the replication inhibitors aphidicolin and L-mimosine (Figure 1B and S1E bottom). Moreover, when the reporter was placed on a plasmid containing the Epstein-Barr virus (EBV) replication origin (oriP) that requires EBNA1 for replication^44–46, g2/Cas9^D10A-induced MMEJ depended on EBNA1 (Figure 1C). These findings support a requirement for replication in converting nicks into DSBs to activate fork-MMEJ.

To confirm DSB formation in the EGFP-MMEJ-AAVS1 reporter after Cas9n cleavage, we modified DSBCapture⁴⁷ using reporter-specific internal primers (Figure S2A). After g2/Cas9^D10A cleavage, DSBs were detected on both sides of the gRNA site, consistent with bidirectional replication at AAVS1 locus, while uncut genomic DNA showed almost no signals (Figure S2B). We also used light-activated Cas9 system (vfCRISPR) with caged gRNA⁴⁸ to induce Cas9n cleavage in G1-arrest cells, and showed that DSBs formed only in cells released from G1 arrest by droplet digital PCR (Figure S2C), confirming that replication is required to convert nicks into DSBs.

MMEJ at broken forks shows asymmetric features.

To assess fork-MMEJ outside of the MMEJ reporter, we used Cas9n to cleave the LBR locus⁴⁹ in human cells with two gRNAs targeting opposite DNA strands (Figure 1D top). Deep sequencing analysis revealed enrichment of indels with MMEJ features (≥ 1bp MH and ≥ 4 bp deletion) after Cas9^D10A and Cas9^H840A cleavage compared to Cas9^WT, with larger deletions and MHs than those from Cas9^WT (Figure 1D, S2D and S2E). Intriguingly, while indels induced by Cas9^WT exhibited a symmetric pattern (Figure 1E), consistent with bidirectional resection at deDSBs (Figure 1F top), those from Cas9n cleavage showed an asymmetric pattern—nicking the top strand leads to deletions extending rightward, whereas nicking the bottom strand resulted in deletions extending leftward (Figure 1E). Cas9n-induced indels were diminished when replication was inhibited by aphidicolin (Figure S2F). Therefore, MMEJ at broken forks (Cas9n) exhibits distinct features compared to cMMEJ at deDSBs (Cas9^WT).

As reported, LBR-g1/Cas9^WT cleavage yielded a prominent +1 bp (1 bp insertion) NHEJ product and two relatively low −7 bp (7 bp deletions with 3 bp MH) cMMEJ products⁴⁹. In contrast, Cas9^D10A and Cas9^H840A cleavage yielded more frequently −7 bp MMEJ products, with +1 bp NHEJ products markedly low (Figure 1G), indicating that fork-MMEJ, rather than NHEJ, is utilized to repair broken forks.

MMEJ induced after Cas9 and Cas9n exhibits different genetic dependence.

Like cMMEJ at deDSBs^18,50–53, Cas9n-induced MMEJ required Polθ and LIG3 (Figure 2A and S3A). However, while RPA suppresses cMMEJ^19,51, RPA2 depletion (Figure 2B and S3B) or degradation (Figure S3C) markedly reduced fork-MMEJ, suggesting that RPA is required for fork-MMEJ. Unlike cMMEJ at deDSBs, which requires MRE11- and CtIP-mediated short end resection to expose MHs^41,54–56, fork-MMEJ (g2/Cas9^D10A) was independent of MRE11 and CtIP and even enhanced by their depletion (Figure 2C and S3D). In addition, fork-MMEJ did not require extensive end resection and was modestly increased upon depletion of BLM, DNA2, and EXO1 as with cMMEJ (Figure S3E). We propose that fork-MMEJ exposes MHs through DNA unwinding rather than end resection, defining a repair pathway distinct from cMMEJ.

We inserted the EGFP-MMEJ reporter into the ROSA locus in mouse embryonic stem (mES) cells, where replication proceeds bidirectionally⁵⁷ (Figure S3F) and found that POLQ-KO strongly reduced both fork-MMEJ and cMMEJ (Figure 2D). POLQ-D2494P/E2495R knock-in mutant affecting the polymerase domain (PolD)⁵³ drastically reduced cMMEJ (g2/Cas9^WT), similar to POLQ-KO, whereas POLQ-K120G helicase domain (HelD)⁵³ mutant exhibited only 30–40% reduction in cMMEJ (Figure 2E left), suggesting that Polθ helicase activity for cMMEJ by removing RPA from ssDNA is important, but not essential¹⁹. However, after g2/Cas9^D10A cleavage, both mutants showed a severe defect in fork-MMEJ, comparable to POLQ-KO (Figure 2E right). Thus, fork-MMEJ relies more on Polθ helicase activity compared to cMMEJ. We speculate that Polθ-HelD is involved in DNA unwinding to expose MHs for fork-MMEJ at seDSBs (Figure 7G right), in addition to its role in antagonizing RPA¹⁹.

(**A-D**) U2OS (EGFP-MMEJ) cells (A, D) or U2OS (EGFP-MMEJ/mCherry-BIR) cells (B, C) treated with or without VE822 (0.5 μM) (A, B), expressing ATR shRNA or shCtrl (C) or expressing RPA2-WT or 4A and 4D mutants with endogenous RPA2 silenced by shRNA (D) were assayed for MMEJ (A, D) or MMEJ and BIR (B, C) after expressing gRNA2 with Cas9^WT or Cas9^D10A. (E) RPE1 or T98G cells treated with VE822 (10 μM, 2 hours) or DMSO were assayed for the ssDNA level with BrdU staining (left) and for cell viability after expressing Polθ shRNA with treatment of VE822 (72 hours) (right). (F) Normal and transformed BJ (ERM) cells were assayed for ssDNA levels (left) and cell viability following Polθ depletion (middle) or ART558 treatment (5 μM, right) in the presence of VE822 (72 hours). (G) Models illustrating the switching control from fork-MMEJ to BIR by ATR activation coupled with end resection (left) and for the fork-MMEJ mechanism at seDSBs upon fork breakage. Data are represented as mean ± SD. See also Figure S4.

Polθ-HelD exhibits activity to unwind DNA in vitro, facilitated by RPA (Figure S3G left)⁵⁸. We further showed that Polθ-HelD unwound DNA duplex, allowing ssDNA to anneal to the template in an RPA-facilitated and ATP-dependent manner (Figure 2F and S3G middle). Polθ-PolD could then utilize the 3’ end of the newly annealed DNA strand to start DNA synthesis, even under conditions requiring strand displacement synthesis, which also required ATP and RPA (Figure 2G and S3G right).

To examine whether Polθ is recruited to seDSBs in cells, we performed chromatin immunoprecipitation (ChIP) analysis after Cas9^D10A cleavage in U2OS (EGFP-MMEJ) cells. Polθ was recruited to seDSBs as efficiently as to deDSBs using γH2AX ChIP to mark DSB formation (Figure S3H), supporting the role of Polθ in fork-MMEJ.

Fork-MMEJ is strongly suppressed by NH tails of 2 nucleotides (nt) or longer 3’ to the MH.

To examine whether MH size influences MMEJ frequency, we generated a set of MMEJ reporters using 2–6 bp MHs for annealing and found that longer MHs promoted higher frequencies of both cMMEJ and fork-MMEJ (Figure S4A). Notably, fork-MMEJ remains relatively more efficient as MH length decreases compared to cMMEJ.

While nicking the top strand of the EGFP-MMEJ-AAVS1 reporter in U2OS cells (g2/Cas9^D10A) induced substantial fork-MMEJ (Figure 1A), nicking the bottom strand (g2/Cas9^H840A) failed to do so (Figure 3A left). Similar results were obtained using the EGFP-MMEJ reporter at the ROSA locus in mES cells (Figure 3A right). Nicking the top or bottom DNA strands would result in fork breakage on leading or lagging strands depending on replication directions (Figure 3B). Four fork breakage types (I-IV, Figure 3B) are predicted at the EGFP-MMEJ reporter after g2/Cas9^D10A (nick top) or g2/Cas9^H840A (nick bottom) cleavage. In a cell population, top-strand nicks produce a mixture of Type I/II, whereas bottom-strand nicks generate a mixture of Type III/IV. Based on the previous studies of fork breakage outcomes³⁶, we speculate that Type II and III breaks on the leading strands generate seDSBs (lead-seDSBs), while replication bypass could occur at Type I and IV breaks on the lagging strands, converting seDSBs to deDSBs (lag-seDSBs/deDSBs) (Figure 3B and S1D).

Besides g2, which cuts 1 bp outside of the left MH, we introduced nicks using gRNAs g4 or g5, 1 bp or 2 bp inside the left MH (Figure 3C). g4 and g5 with Cas9^H840A (nick top, I+II) but not with Cas9^D10A (nick bottom, III+IV) induced efficient fork-MMEJ, supporting efficient fork-MMEJ at Type I and/or II, but not at Type III/IV fork breakages. Type I/II seDSBs generated by g2, g4, or g5 commonly have short (0–1 nt) 3′ NH tails at the priming ends for fork-MMEJ DNA synthesis, whereas Type III/IV seDSBs retain long (17–20 nt) 3′ NH tails (Figure 3B). We speculate that long 3′ NH tails outside the MH at seDSBs on the broken parental strands block fork-MMEJ DNA synthesis (Figure S4B). Using g1/Cas9^H840A to generate Type I/II seDSBs with 7 nt 3’ NH tails resulted in inefficient fork-MMEJ at the level comparable to g1/Cas9^D10A-induced Type III/IV breaks with 11-nt tails (Figure 3D and 3B), further supporting that long 3′ NH tails inhibit fork-MMEJ.

We also designed a new EGFP-MMEJ-5879 reporter where 4 gRNAs generate no (0 nt), 1 nt, 2 nt or 3 nt NH tails to the left MH (Figure 3E top). Cas9^WT induced comparable cMMEJ at all gRNA sites (Figure 3E bottom), indicating similar cutting and cMMEJ efficiency regardless of the 3’ NH tail length. In contrast, Cas9^D10A induced efficient fork-MMEJ (nick top, Type I+II) only with 0–1 nt NH tails (higher efficiency at 0 bp), but not with ≥2 nt NH tails (Figure 3E bottom). Thus, 3’ NH tails (≥2 nt) outside of MH, block fork-MMEJ at seDSBs but not cMMEJ at deDSBs (Figure S4B). We propose that while cMMEJ involves NH tail trimming from both DSB ends (Figure S1A), Polθ may directly initiate DNA synthesis during fork-MMEJ from the 3’ broken parental strand end without trimming, allowing up to 1 nt NH tail (Figure S4B, S4C and 7G step 3). Consistently, previous biochemical analysis showed that Polθ can tolerate one unpaired base at the 3’ ends for DNA synthesis in vitro⁵⁹. We further demonstrated that while 1- nt NH tail could be tolerated with reduced efficiency, a 2-nt NH tail completely blocked DNA synthesis (Figure S4D).

Fork-MMEJ is used more frequently at seDSBs on the leading strands induced by Cas9n.

In non-B cells, including mES cells, origins are silenced in the ~400 kb temporal transition region (TTR) at the Igh locus, resulting in unidirectional replication by a single fork⁶⁰. Accordingly, Igh-g2/Cas9^D10A cleavage generates leading-strand breakage, producing lead-seDSBs on the left side of the cleavage site. Site-specific DSBCapture (Figure S2A) detected DSBs predominantly on the left side, whereas Igh-g2/Cas9^WT cleavage produced DSBs on both sides (deDSB) (Figure S5A). These data support seDSB formation after Cas9n cleavage.

We inserted the EGFP-MMEJ reporter in both orientations at Igh TTR (Igh-MMEJ-For and Igh-MMEJ-Rev, Figure 4A) in mES cells, without influencing replication direction in that region (Figure S5B). As replication is unidirectional at Igh TTR, we can distinguish four types of seDSBs after g2/Cas9^D10A and g2/Cas9^H840A cleavage (Figure 4B). g2/Cas9^WT induced similar levels of cMMEJ in both Igh-MMEJ-For and Igh-MMEJ-Rev reporters, comparable to that in the ROSA-MMEJ reporter (Figure 4C left), suggesting that replication direction relative to the MMEJ inserted orientation does not influence cMMEJ at deDSBs. However, fork-MMEJ was more efficiently induced at Type II seDSBs on leading strands (Igh-MMEJ-Rev, g2/Cas9^D10A, lead-seDSBs) compared with Type I seDSBs/deDSBs on lagging strands (Igh-MMEJ-For, g2/Cas9^D10A, lag-seDSBs/deDSBs) (Figure 4C right). As expected, fork-MMEJ was inefficient at Type III seDSBs and IV seDSBs/deDSBs (ROSA-MMEJ, Igh-MMEJ-For and Igh-MMEJ-Rev, g2/Cas9^H840A), which carry long 3’ NH tails (Figure 4C right and 4B). We also used another gRNA, g6, which cleaves within the right MH, and obtained similar results that only Type II lead-seDSBs (Igh-MMEJ-For, g6/Cas9^D10A) were efficiently repaired by fork-MMEJ (Figure 4D and S5C). These results suggest that fork-MMEJ is favored on broken leading strands after Cas9n cleavage.

In Xenopus extracts, nickase-induced lag-seDSBs contain ~70 nt 3’ ssDNA overhangs, whereas lead-seDSBs are blunt or have up to 3 nt 5′ overhangs³⁸. The pre-existing 3’ ssDNA overhangs on broken lagging strands (Figure S1D) may preferentially channel seDSB repair to BIR, thereby limiting fork-MMEJ (Figure S7D). Since replication at Igh TTR is unidirectional⁶⁰, efficient fork-MMEJ at Type II seDSBs supports the model that fork-MMEJ directly engages seDSBs for repair, preferentially on the leading strands and without requiring fork convergence.

We also analyzed indel formation directly at the Igh TTR locus in mES cells, using a pair of gRNAs, Igh-g2/Cas9^D10A and Igh-g4/Cas9^D10A, which cleave the bottom (leading strand) and top (lagging strand) strands, respectively (Figure 4E top). While indel frequency with MMEJ features (≥ 1bp MH and ≥ 4 bp deletion) at lead-seDSBs (Igh-g2/Cas9^D10A) was significantly higher than that at lag-seDSBs/deDSBs (Igh-g4/Cas9^D10A), indel frequency at deDSBs (Igh-g2/Cas9^WT, Igh-g4/Cas9^WT) was comparable (Figure 4E bottom). In addition, we analyzed unidirectionally replicated genomic loci in U2OS cells (Figure S5D top) using pairs of gRNAs/Cas9^D10A to cleave either the leading or lagging strands (Figure S5D bottom left), and the frequency of indels with MMEJ features (≥ 1bp MH and ≥ 4 bp deletion) at those loci was higher on the leading strands (Figure S5D bottom right). These findings are consistent with our MMEJ reporter results, showing that fork-MMEJ operates preferentially at lead-seDSBs (Figure 4C, 4D, Type II).

Moreover, at the Igh locus in mES cells, Cas9^WT-induced deDSBs produced symmetric deletions (Figure 4F left), whereas Cas9^D10A-induced seDSBs yielded asymmetric deletions (Figure 4F middle), as observed at the LBR locus in U2OS cells (Figure 1E). As expected, Cas9n-induced indels on the leading strands at the Igh locus (Igh-g2/Cas9^D10A) were reduced when replication was inhibited by aphidicolin (Figure S5E). Collectively, these data support our model that the 3’ end of the broken parental strand searches for internal MH on the other end and directly primes DNA synthesis without end trimming (Figure 4F right and S4C).

Fork-MMEJ functions together with BIR to repair seDSBs

MMEJ is error prone^18,19, it can act faster than HR/BIR, requiring only short (cMMEJ)⁴¹ or no (fork-MMEJ, this study) end resection. To compare MMEJ and BIR at broken forks, we established an EGFP-MMEJ/mCherry-BIR reporter, simultaneously monitoring MMEJ and BIR. We inserted the BIR donor cassette EG-T2A-mCherry upstream of the EGFP-MMEJ cassette (Figure 5A top). When MMEJ is used, green cells are produced. To initiate BIR, the EG in the EGFP-MMEJ cassette invades the EG homology in the EG-T2A-mCherry BIR donor cassette on its sister chromatid, followed by replicating 2 kb through mCherry and reaching the FP homology in the EGFP-MMEJ cassette to complete BIR/LTGC. As a result, the CMV promoter is placed in front of the EG-T2A-mCherry cassette, producing red cells (Figure 5A bottom, BIR/LTGC).

Figure 5. — (A) Schematic drawings of the EGFP-MMEJ/mCherry-BIR reporter and the repair products by cMMEJ and BIR after Cas9^WT cleavage. (B) MMEJ or BIR was scored in U2OS (EGFP-MMEJ/mCherry-BIR) cells by FACS to determine EGFP or mCherry positive cells after cleavage by gRNA2 with Cas9^WT and Cas9^D10A. (C) Time course analysis of MMEJ and BIR in U2OS (EGFP-MMEJ/mCherry-BIR) cells after gRNA2 cleavage with Cas9^WT and Cas9^D10A. (D-F) MMEJ and BIR assays in U2OS (EGFP-MMEJ/mCherry-BIR) cells expressing shRNAs for CtIP (D), MRE11 (D), PIF1(E) or Polθ (F) after gRNA2 cleavage with Cas9 and Cas9n. Data are represented as mean ± SD. See also Figure S6.

The EGFP-MMEJ/mCherry-BIR reporter was integrated into the AAVS1 locus in U2OS cells. cMMEJ/fork-MMEJ (green) and BIR (red) were used concurrently after g2/Cas9^WT and g2/Cas9^D10A cleavage (Figure 5B), whereas g2/Cas9^H840A induced BIR, but not fork-MMEJ (Figure 5B), consistent with long NH tails blocking fork-MMEJ (Figure 3A). Time-course experiments showed that cMMEJ (g2/Cas9^WT) and fork-MMEJ (g2/Cas9^D10A) were initiated prior to BIR, with substantial BIR observed at later time points (Figure 5C), supporting the quick launch of MMEJ over BIR at both deDSBs and seDSBs. Consistent with MRE11 and CtIP suppressing fork-MMEJ (Figure 2C), their depletion impaired BIR but increased fork-MMEJ at seDSBs (g2/Cas9^D10A), while both cMMEJ and BIR at deDSBs (g2/Cas9^WT) decreased (Figure 5D and S6A). We propose that while fork-MMEJ acts prior to end resection to repair seDSBs, the onset of end resection suppresses fork-MMEJ and promotes BIR (Figure 7G left).

To test whether fork-MMEJ/cMMEJ and BIR can compensate for each other, we depleted PIF1 or Polθ by shRNAs in U2OS (MMEJ/BIR) cells (Figure S6B). PIF1 loss impaired BIR at both deDSBs (g2/Cas9^WT) and seDSBs (g2/Cas9^D10A), with little effect on cMMEJ (g2/Cas9^WT) and a small increase in fork-MMEJ (g2/Cas9^D10A) (Figure 5E). Conversely, depletion of Polθ resulted in a strong defect in cMMEJ and fork-MMEJ, but a small (g2/Cas9^D10A) or no (g2/Cas9^WT) increase in BIR (Figure 5F). These data suggest that despite being used simultaneously, MMEJ and BIR are not completely interchangeable. Accordingly, while depletion of Polθ or PIF1 alone caused hydroxyurea (HU) sensitivity (Figure 6A) and impaired replication restart efficiency and speed (Figure 6B)^23,61, combined inactivation produced more severe defects (Figure 6A, 6B, S6C and S6D).

Figure 6. — (A) U2OS cells expressing shRNAs for Polθ, PIF1 or both were treated with HU (72 hours), and cell viability was determined. (B) DNA fiber analysis was performed in WT or *PIF1* KO U2OS cells expressing Polθ shRNA or shCtrl. Representative restarted forks are shown (left). The percentage of restarted/stalled forks and new origin firing (middle), and restarted fork speed (right) are determined. (C) PLA of HA-Polθ and PCNA in U2OS cells with or without HU treatment (2 mM, 24 hours). (D) PLA showing colocalization of HA-Polθ and PCNA with or without PIF1 depletion (left) and of Flag-PIF1 and PCNA with or without Polθ depletion (right), before and after HU treatment (2 mM, 24 hours). Scale bar, 2 μm. Data are represented as mean ± SD. See also Figure S6.

PCNA interacts with Polθ and PIF1 and their interactions are increased upon PCNA ubiquitination^62,63 (Figure S6E). In situ proximity ligation assay (PLA) revealed that HU enhanced Polθ-PCNA interaction (Figure 6C), which further increased after PIF1 depletion (Figure 6D top and S6B left), and vice versa for PIF1-PCNA interaction after Polθ depletion (Figure 6D bottom and S6B right). Furthermore, HU-induced recruitment of Polθ and PIF1 to γH2AX-marked DSBs required PCNA (Figure S6F and S6G) and its ubiquitination (Figure S6H). Together, these data suggest that PCNA recruits Polθ and PIF1 to broken forks through direct interactions, promoting fork-MMEJ and BIR.

ATR suppresses fork-MMEJ and shifts repair toward BIR in conjunction with end resection.

While ATR promotes HR⁶⁴, its role in MMEJ remains unclear. Strikingly, ATR inhibition (VE822) drastically increased fork-MMEJ, but not cMMEJ in U2OS (EGFP-MMEJ-AAVS1) cells (Figure 7A). Moreover, ATR inhibition (EV822) or depletion (shRNA) in U2OS (MMEJ/BIR) cells reduced BIR at both deDSBs (Cas9^WT) and seDSBs (Cas9^D10A), whereas cMMEJ (Cas9^WT) was unaffected and fork-MMEJ (Cas9^D10A) was significantly increased (Figure 7B, 7C and S4E). These results suggest that ATR is required for BIR at both deDSBs and seDSBs, but specifically suppresses fork-MMEJ upon fork breakage, without affecting cMMEJ at deDSBs.

RPA suppresses cMMEJ but is required for fork-MMEJ (Figure 2B). Notably, RPA2 is progressively phosphorylated by ATR following end resection at replication-associated, but not replication-independent DSBs⁶⁵. Strikingly, in the RPA2-S4A/S8A/T21A/S33A mutant (RPA2–4A) cells, in which the key phosphorylation sites (S4 and S8: DNAPK dependent^66–68 and ATR partially dependent⁶⁵; T21 and S33: ATR dependent⁶⁵) were mutated and endogenous RPA2 was depleted, fork-MMEJ but not cMMEJ was significantly increased (Figure 7D and S4F). Conversely, the phosphomimetic RPA2-S4D/S8D/T21D/S33D mutant (RPA2–4D) was defective in fork-MMEJ but not cMMEJ (Figure 7D and S4F). We propose that as end resection progresses at seDSBs on broken forks, ATR is activated to phosphorylate RPA2, which impairs RPA activity required for fork-MMEJ, thereby suppressing fork-MMEJ (Figure 7G left).

To test whether MRE11-mediated end resection, ATR activation, and RPA phosphorylation act in the same pathway to suppress fork-MMEJ, we treated MRE11-depleted or RPA2–4A (+RPA2 shRNA) cells with ATR inhibitor (ATRi). ATR inhibition markedly increased fork-MMEJ in controls but had little effect in MRE11-depleted or RPA2–4A cells (Figure S4G). Using human nuclear extracts⁶⁹, we further showed that ~50 nt ssDNA overhangs were sufficient to trigger ATR-mediated RPA2 phosphorylation, with stronger induction by longer overhangs (Figure S4H).

Inhibition of Polθ and ATR shows a synergistic effect to eradicate cancer cells under replication stress

ATR protects replication forks^5,70, preventing DSBs⁷¹. Consistent with ATR deficiency reducing BIR and increasing fork-MMEJ, co-inhibition of ATR and Polθ synergistically killed T98G tumor cells but, interestingly, not untransformed RPE1 cells (Figure 7E right and S4I). As described, ATR inhibition leads to ssDNA accumulation, but only excessive ssDNA levels result in DSB formation⁷¹. We speculate that in normal cells, where RS is low, ATRi-induced ssDNA and DSBs remain minimal and tolerable, but in tumor cells with high RS, ATRi drives ssDNA beyond the threshold, generating DSBs⁷¹ (Figure S7F). Consistently, T98G, but not RPE1 cells, exhibited high RS, as indicated by ssDNA accumulation after short ATRi exposure⁷¹ (Figure 7E left). Similarly, oncogene-transformed BJ cells [E1A, H-RAS-V12, MDM2 (ERM)], but not isogenic normal BJ cells, displayed high RS (Figure 7F, left). In contrast to normal BJ cells, while ATRi alone (VE822) caused a small increase of cell death in BJ-ERM cells, combining ATRi with Polθ depletion or inhibition induced synergistic lethality (Figure 7F middle and right, and S4J). These results suggest that combined inhibition of Polθ and ATR selectively eradicates replication-stressed tumor cells while sparing normal cells.

Discussion

The mechanism underlying fork-MMEJ differs from cMMEJ at deDSBs.

Fork-MMEJ on broken forks differs from cMMEJ at deDSBs in that it does not require end resection but relies on RPA. We propose that Polθ helicase activity promotes DNA unwinding to expose microhomologies for repair (Figure 7G, right). In vitro, Polθ-HelD unwinds DNA, which is enhanced by RPA⁵⁸, and we showed that this activity allows ssDNA annealing and priming of DNA synthesis by Polθ-HelD. Since no direct interaction between RPA and Polθ-HelD or Polθ-PolD was detected (Figure S7A), the requirement for RPA in fork-MMEJ may stem from its role in stabilizing ssDNA during Polθ-HelD-mediated unwinding. Given its weak helicase activity⁵⁸, Polθ-HelD may cooperate with other helicases to promote DNA unwinding, with these activities requiring RPA.

Another notable difference is that cMMEJ tolerates long NH tails (flaps) at both ends of a DSB, but fork-MMEJ is strongly suppressed by ≥2 nt NH tails at the 3′ end of the broken parental strand (Type III and IV: Figure 3B, blue circle of the 3’ end; Figure S4B), while long NH tails are tolerated at the 5′ end (Type I and II: Figure 3B, blue rectangle of the 5’ end; Figure S4B). Upon fork breakage, the two broken ends on the parental strands need to religate for restoring fork integrity (Figure S4C). This requires the 3’ end of the broken parental strand to anneal with the 3’ daughter strand of the other end (lead-seDSBs) or the other unbroken parental strand (lag-seDSBs) using an MH sequence (Figure S4C). We propose that the MH at the 3’ end of the broken parental strand is used to search for MH internal of the seDSB (designated as 3’ MH-searching end, Figure S4C). This is supported by asymmetric deletion patterns following Cas9n cleavage (Figure 1E, 1F bottom and 4F), in contrast to the symmetric deletions observed in cMMEJ at deDSBs resulting from bidirectional end resection (Figure 1F top). The tolerance for a 1-nt, but not a 2-nt NH tail at the 3’ MH-searching end for fork-MMEJ is consistent with the biochemical activity of Polθ, showing that only a single unpaired nucleotide, but not more than one, at the 3’ end permits Polθ-PolD-mediated DNA synthesis in vitro⁵⁹ (Figure S4D).

We propose that fork-MMEJ prioritizes speed by using DNA unwinding rather than time-consuming end resection, directly engaging the 3′ end of the broken parental strand for microhomology searching and DNA synthesis. In contrast to deDSBs, where c-NHEJ acts immediately and cMMEJ follows end resection, c-NHEJ is unavailable at seDSBs on broken forks, necessitating rapid repair by fork-MMEJ. In addition, since MH annealing is transient and prone to dissociation, initiating bridging DNA synthesis directly from the 3′ end without trimming can quickly stabilize broken forks for efficient repair (Figure 7G, step 3). In cMMEJ, Polδ uses its exonuclease activity to remove NH tails⁷². Because this process requires sequential handoffs—Polθ aligns MHs, then a switch to Polδ for NH removal with each round to remove one nucleotide, followed by a switch back to Polθ for MH searching⁷³—skipping trimming could accelerate fork-MMEJ.

Using our reporter, we score a fixed event with designed MHs to produce green cells; in this context, 3′ NH tails, if present on the searching strand ends would inhibit fork-MMEJ (Figure S7B left). However, at genomic sites, the 3’ MH-searching end can search a broader region for MHs on the opposing end (Figure S7B right). Since there is more than a 93% chance of finding a ≥ 3bp MH within 15 bp of any given pair of DNA ends, productive fork-MMEJ is highly likely for a fixed end to find ≥1–3 bp MHs at the other DSB end⁷⁴.

A proposed working model of fork-MMEJ for seDSB repair.

We propose a working model as illustrated in Figure 7G. Upon fork breakage, the two Polθ-HelDs tether the broken forks through dimerization^75,76, with one binding to the seDSB and the other to the unreplicated DNA duplex with a 3’ broken end (Figure 7G right top). Both DNA duplex of the unreplicated DNA and the other seDSB end (lead-seDSB; lag-seDSB if ssDNA overhang is not sufficient) need to be unwound to expose MHs (Figure 7G, step 1 and S4C). Subsequently, the 3’ end on the broken parental strands (3’ MH-searching end) is directly used to search for an MH internal of the other end and anneals with it (Figure 7G, step 2 and S4C), followed by DNA synthesis from the 3’ end without trimming (Figure 7G, step 3). After bridging DNA synthesis from one end, the broken forks are stably tethered together. In contrast to the 3’ MH-searching end that is directly used for priming DNA synthesis, the second 3’ end at lead-seDSB requires Polδ exonuclease activity⁷² and/or 3’-flap nucleases, such as XPF/ERCC1⁷⁷, to remove the 3’ flap prior to initiating DNA synthesis (Figure 7G, step 4). In addition, strand displacement DNA synthesis may occur, generating 5’ flaps which need to be removed by nucleases at both lead-seDSB and lag-deDSB (Figure 7G, step 4). 5’-flap nucleases such as SLX1^77,78, DNA2⁷⁹, FEN1⁸⁰ and FAN1⁸¹ could possibly contribute, which requires further investigation. Polθ is not a processive polymerase, and a switch from Polθ to Polδ⁷² may occur during strand displacement DNA synthesis. In addition, since Polε disassociates from CMG at lead-seDSBs^36,38, it needs to reload on the leading strand after fork-MMEJ to restore replication and restart forks (Figure 7G, step 6). Fork-MMEJ is completed by end ligation (Figure 7G right, step 5).

Fork-MMEJ directly acts on seDSBs, alongside BIR, to repair replication-associated fork breakage.

While BIR is a well-established mechanism for repairing seDSBs, we found that fork-MMEJ can directly repair seDSBs immediately upon fork breakage, prior to BIR engagement. Both Polθ and PIF1 are recruited by ubiquitinated PCNA upon RS, but pathway choice is not determined merely by their recruitment, rather by the extent of 3′ end resection, which governs the balance between fork-MMEJ and BIR. Using the dual MMEJ/BIR reporter, we showed that inhibiting end resection by depleting MRE11 or CtIP strongly stimulates fork-MMEJ while suppressing BIR, whereas depleting PIF1, which functions downstream of resection, blocks BIR but only modestly increases fork-MMEJ. Thus, while end resection controls the choice between fork-MMEJ and BIR, once committed, the two pathways cannot fully compensate for each other. Consistently, simultaneous inactivation of both pathways causes stronger defects in fork restart and hypersensitivity to RS than loss of either alone.

Recent studies have shown that in addition to BIR, HR is also employed at broken forks^82,83, following the formation of deDSBs after fork convergence and replication bypass (Figure S7C). To assess HR involvement, we established a competition reporter to monitor fork-MMEJ and HR at broken forks (Figure S7D top). While both cMMEJ and fork-MMEJ occurred prior to HR (Figure S7D bottom), HR increased at later time points, with its usage eventually slightly exceeding cMMEJ (Cas9^WT), but remaining lower than fork-MMEJ (Cas9^D10A). This differs from BIR after Cas9^D10A cleavage, which increased to levels comparable to fork-MMEJ at later time points (Figure 5C). These findings support the model that seDSBs at broken forks are first repaired by fork-MMEJ, followed by BIR, with HR engaged when seDSBs are converted to deDSBs (Figure S7C). They also indicate that BIR is used more frequently than HR at broken forks, consistent with our previous observations⁶¹.

Using the EGFP-MMEJ reporter, we found that fork-MMEJ is more preferentially used on leading strands after Cas9n cleavage, consistent with higher indel accumulation on leading strands at genomic sites exhibiting unidirectional replication over ~100 kb (Figure S7E). This bias likely reflects the presence of 3′ ssDNA overhangs (~70 nt) at lag-seDSBs³⁸, which could directly channel repair toward BIR, whereas lead-seDSBs are largely blunt-ended and require resection to initiate BIR (Figure S7C). Rapid conversion of lag-seDSBs to deDSBs by CMG bypass may also accelerate HR on lagging strands, further reducing fork-MMEJ usage.

MMEJ is generally viewed as a backup DSB repair pathway when HR is defective^13,23. BIR also requires key HR factors such as RAD51 and BRCA1⁶¹. In RAD51-deficient cells, seDSBs are converted to hyper-resected deDSBs as revealed by End-seq analysis³⁶, and exposed MHs likely would channel repair toward cMMEJ rather than fork-MMEJ. Furthermore, Polθ-mediated fork-MMEJ is distinct from previously described Polθ activities of gap-filling ²⁴ and microhomology-mediated gap skipping²⁵ at stalled forks, which occur before DSB formation. In contrast, fork-MMEJ functions after DSB formation at broken forks and is linked to the end-joining activity of Polθ in MMEJ.

ATR drives the transition of fork-MMEJ to BIR.

Fork-MMEJ provides rapid repair of seDSBs on broken forks but needs to be tightly regulated given its error-prone nature, and our study suggests that ATR plays a critical role in controlling the use of fork-MMEJ. At replication-associated DSBs, end resection-generated RPA-ssDNA drives ATR-mediated RPA2 phosphorylation, coupling resection extent to RPA2 phosphorylation⁶⁵. Our model is that progressive resection at replication-associated DSBs activates ATR to phosphorylate RPA2, which in turn suppresses fork-MMEJ specifically without affecting cMMEJ at deDSBs (Figure 7G left). It remains possible that other ATR targets, in addition to RPA2, may contribute to fork-MMEJ suppression.

ATR inhibition suppresses BIR but causes a pronounced increase in fork-MMEJ, implying that ATR inhibition would result in a strong reliance on fork-MMEJ for repairing seDSBs. Indeed, simultaneous inhibition of ATR and Polθ synergistically kills cancer cells by blocking both repair pathways. In normal cells, RS is low, and even with ATR inhibition, ssDNA levels remain tolerable with minimal DSB formation (Figure S7F), resulting in a low demand for fork repair. In contrast, cancer cells experience high RS⁷¹, and ATR inhibition therefore causes extensive ssDNA accumulation, fork breakage, and seDSBs, which require fork-MMEJ and BIR for repair. Consequently, combined ATR and Polθ inhibition effectively eradicates cancer cells with low toxicity to normal cells, providing a new strategy for targeted cancer therapy.

Limitations of the study

One technical limitation is that currently there are no reliable assays to specifically measure DNA unwinding by helicases in cells; therefore, the helicase activity of Polθ was assessed only using in vitro biochemical analyses in our study. In addition, building on the mechanistic understanding gained from the studies using the EGFP-MMEJ reporter, we validated the general principles through direct deep-sequencing analysis of indels generated at genomic loci following Cas9 or Cas9n cleavage. For indel analysis at each cleavage site, we need to analyze at least 50K reads for accurate measurements. This limits our study to examining sites one by one, rather than using whole-genome deep sequencing to assess many sites simultaneously.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Xiaohua Wu (xiaohwu@scripps.edu).

Materials availability

Materials generated in this study are available through lead contact upon request with a standard Material Transfer Agreement.

Data and code availability

Raw sequencing data (site-specific sequencing, nascent strand sequencing, Repli-seq) are available in the NIH Sequence Read Archive (SRA) as BioProject: PRJNA1235770. The original uncropped images are deposited at Mendeley Data: 10.17632/d79jttdxyc.4. The accession numbers of the published nascent strand sequencing of mouse embryonic stem cells are indicated in the Key Resources Table.
This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Key Resources Table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
mouse Anti- BrdU	BD Biosciences	Cat# 555627, RRID: AB_395993
rat anti-BrdU	Abcam	Cat# ab6326
mouse anti-BrdU	BD Biosciences	Cat# 347580, RRID: AB_400326
Rabbit anti-MRE11	Cell Signaling Technology	Cat# 4895
Rabbit anti-CtIP	Proteintech	Cat# 12624–1-AP, RRID: AB_2175250
Mouse anti-RPA1	Sigma-Aldrich	Cat# NA13
Rabbit anti-RPA2	Bethyl	Cat# A300–244A
Rabbit anti-PCNA	Cell Signaling Technology	Cat# 13110
Rabbit anti-MCM2	Proteintech	Cat# 10513–1-AP, RRID: AB_2142131
Rabbit anti-DNA2	Proteintech	Cat# 18727–1-AP, RRID: AB_10637998
Mouse anti-KU70	Santa Cruz Biotechnology	Cat# sc-17789
Mouse anti-HA	Santa Cruz Biotechnology	Cat# sc-7392
Mouse anti-Flag	Sigma-Aldrich	Cat# F1804
Rabbit anti-BLM	Bethyl	Cat# A300–110A
Rabbit anti-EXO1	Bethyl	Cat# A302–640A
Mouse anti-ATR	Santa Cruz Biotechnology	Cat# sc-515173
Rabbit anti-LIG3	Proteintech	Cat# 26583–1-AP, RRID: AB_2880562
Rabbit anti-β-Actin	Proteintech	Cat# 20536–1-AP, RRID: AB_10700003
Rabbit anti-γH2AX	Sigma-Aldrich	Cat# 07–164
Rabbit anti-Polθ	Cell Signaling Technology	Cat# 64708
Rabbit anti-PIF1	Affinity Bioscience	Cat# DF9256
Peroxidase AffiniPure Goat Anti-Mouse IgG(H+L)	Jackson ImmunoResearch Labs	Cat# 115–035-146, RRID: AB_2307392
Peroxidase AffiniPure Goat Anti-Rabbit IgG(H+L)	Jackson ImmunoResearch Labs	Cat# 111–035-144, RRID: AB_2307391
Alexa 594 Goat anti-rat IgG(H+L)	Invitrogen	Cat# A11007
Alexa 488 Goat anti-mouse IgG(H+L)	Invitrogen	Cat# A11029
Rabbit anti-Phospho RPA32 (S33)	Bethyl	Cat# BLR363N
Bacterial and virus strains
E. coli Rosetta 2 (DE3)	Novagen	Cat# 71397
Chemicals, peptides, and recombinant proteins
dTAG-13	MCE	Cat# HY-114421
BrdU	Sigma-Aldrich	Cat# B5002
1×Halt^™ Protease and Phosphatase Inhibitor	Thermo Scientific	Cat# 78440
streptavidin	Invitrogen	Cat# 434301
proteinase K	NEB	Cat# P8107
CldU	Sigma-Aldrich	Cat# C6891
IdU	Sigma-Aldrich	Cat# I7125
Doxycycline	MCE	Cat# HY-N0565
Mirin	MCE	Cat# HY-19959
ART558	MCE	Cat# HY-141520
VE822	MCE	Cat# HY-13902
Palbociclib	MCE	Cat# HY-50767
cOmplete proteinase inhibitors	Roche	Cat# 11697498001
T4 DNA polymerase	NEB	Cat# M0203
T4 PNK	NEB	Cat# M0201
T4 DNA ligase	NEB	Cat# M0202
Alt-R^™ S.p. Cas9 D10A Nickase V3	IDT	Cat# 1081062
PreScission protease (HRV3C) recombinant protein	Abnova	Cat# P7352
ProLong Gold antifade mountant	Invitrogen	Cat# P10144
Critical commercial assays
NEBNext® Ultra^™ II FS DNA Library Prep Kit for Illumina	NEB	Cat# E7805
QuikChange site-directed mutagenesis kit	Agilent	Cat# 200514
Glutathione sepharose resin	Clontech	Cat# 635607
Cell Counting Kit-8 (CCK-8)	Dojindo	Cat# CK04
Protein G dynabeads	Invitrogen	Cat# 10003D
iScript cDNA Synthesis Kit	Bio-Rad	Cat# 1708890
Luna Universal qPCR Master Mix	NEB	Cat# M3003
Duolink® In Situ Detection Reagents Red	Sigma-Aldrich	Cat# DUO92008
Duolink® In Situ Detection Reagents Green	Sigma-Aldrich	Cat# DUO92014
DNeasy Blood & Tissue Kits	QIAGEN	Cat# 69504
SE Cell line 4D-NucleofectorTM X kit	Lonza	Cat# V4XC-1032
ddPCRTM Supermix for Probes (No dUTP)	Bio-Rad	Cat# 186–3023
Lipofectamine® 2000 Transfection Reagent	Invitrogen	Cat# 11668019
NEBNext® Multiplex Oligos for Illumina	NEB	Cat# E6448
Deposited data
Raw data of site-specific PCR deep sequencing, Nascent strand sequencing of U2OS cells, Repli-seq data of mouse Embryonic stem cells	This paper	NIH Sequence Read Archive: PRJNA1235770
Original images of uncropped gels	This paper	Mendeley Data: 10.17632/d79jttdxyc.4
Nascent strand sequencing of mouse Embryonic stem cells	⁵⁷	GEO: GSM3814167, GSM3814168, GSE131699
Experimental models: Cell lines
Human: 293T	ATCC	CRL-3216
Human: U2OS	ATCC	HTB-96
Human: T98G	ATCC	CRL-1690
Human: RPE1(hTERT RPE-1)	ATCC	CRL-4000
Human: BJ	ATCC	CRL-2522
E14(ES-E14TG2a)	ATCC	CRL-1821
Oligonucleotides
Alt-R^™ CRISPR-Cas9 tracrRNA	IDT	Cat# 1072532
Oligos used as DNA substrate and PCR primers	Table S2	N/A
Recombinant DNA
EGFP-MMEJ-AAVS1 plasmid	This paper	N/A
EGFP-MMEJ/mCherry-BIR plasmid	This paper	N/A
mCherry-MMEJ/EGFP-mCherry-HR plasmid	This paper	N/A
pCDH-CMV	Addgene	# 72265
pCDH-CMV-ISceI-Puro	⁸⁷	N/A
pCDH-CMV-Flag-RPA2-Neo	This paper	N/A
pCDH-CMV-Flag-RPA2(S4A/S8A/T21A/S33A)-Neo	This paper	N/A
pCDH-CMV-Flag-RPA2(S4D/S8D/T21D/S33D)-Neo	This paper	N/A
pCDH-CMV-Flag-PIF1-Neo	This paper	N/A
pCDH-CMV-HA-RPA2-FKBP12^F36V	This paper	N/A
pCDNA-HA-Polθ-Neo	This paper	N/A
Tet-On 3×Flag Polθ	Dr. Dale A. Ramsden	N/A
pCEP4	NovoPro	Cat# V012686
EBV/oriP-EGFP-MMEJ plasmid	This paper	N/A
EBV/oriP-EGFP-MMEJ plasmid (no EBNA1)	This paper	N/A
pCW-Cas9	Addgene	# 50661
pCW-Cas9^D10A	This paper	N/A
pLKO.1-blast	Addgene	# 26655
pMD2.G	Addgene	# 12259
psPAX2	Addgene	# 12260
pSpCas9(BB)-2A-Puro (PX459) V2.0	Addgene	# 62988
lentiCRISPR v2	Addgene	# 52961
lentiCRISPR v2-D10A	This paper	N/A
lentiCRISPR v2-H840A	This paper	N/A
pCDH-EF1-FHC-POLQ	Addgene	# 64875
Sumo3 PolQM1	Addgene	# 78462
pSumo3-Polθ-HelD	This paper	N/A
pSumo3-Polθ-HelD(K121A)	This paper	N/A
pET28a-RPA1/RPA2/RPA3	This paper	N/A
Software and algorithms
ImageJ	⁸⁴	https://imagej.net/ij/
GraphPad Prism10	GraphPad	https://www.graphpad.com/
CellProfiler 4.2.6	CellProfiler	https://cellprofiler.org/
FlowJo v10	BD	https://www.flowjo.com/

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STAR Methods

Experimental model and study participant details

Cell lines and cell culture

U2OS (human osteosarcoma), HEK293T (human embryonic kidney), and T98G (human glioblastoma) and RPE1 (human retina) were obtained from ATCC and tested negative for mycoplasma contamination. BJ (ERM) tumor cells were generated by infecting BJ cells with retroviruses encoding oncogenes E1A, H-RAS-V12 (RAS) and MDM2 (ERM).

U2OS, HEK293T, BJ cells are grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, GeminiBio) and 1% penicillin-streptomycin containing glutamine (Gibco). T98G cells are grown in Minimum essential medium (MEM, Gibco) supplemented with 10% fetal bovine serum (FBS, GeminiBio), 1% non-essential amino acids (NEAA, Gibco) and 1% penicillin-streptomycin containing glutamine (Gibco). RPE1 cells are grown in DMEM/F-12 Medium supplemented with 10% fetal bovine serum (FBS, GeminiBio) and 1% non-essential amino acids (NEAA, Gibco). E14 mouse embryonic stem (mES) cells are grown in Glasgow modification of Eagle’s medium (GMEM, Sigma) supplemented with 15% ES-qualified FBS (Gibco), 1% non-essential amino acids (Gibco), 2 mM L-glutamine (Life Technologies), 0.1 mM β-mercaptoethanol (Sigma), 1mM sodium pyruvate (Gibco) and leukemia inhibitory factor (LIF) (2000U/ml, Amsbio). All cells are cultured at 37°C in 5% CO₂ atmosphere.

Method details

Plasmid construction and generation of the reporter cell lines

The CMV-EGFP-MMEJ reporter was generated by placing a disrupted full-length EGFP under the control of a CMV promoter, with insertion of an 18-bp sequence containing in-frame stop codons and a 9-bp microhomology sequence between E112 and V113 as described⁸⁵.

The EGFP-MMEJ/mCherry-BIR reporter was generated by placing the sequence encoding the 1–108 amino acid of EGFP with an in-frame fusion of T2A, followed by a full-length mCherry, in front of the EGFP-MMEJ cassette as shown in Figure 5A.

To generate the mCherry-MMEJ/EGFP-mCherry-HR reporter, the MMEJ cassette was constructed by inserting into the EGFP open reading frame a gRNA cleavage site followed by three stop codons, flanked by a duplication of 9-bp microhomology sequence that replaced the chromophore of EGFP (T65/Y66/G67)⁸⁶. An in-frame T2A-mCherry sequence was fused to the 3’ of the MMEJ cassette. A truncated EGFP fragment (1–167 aa) was placed in front of the MMEJ cassette to serve as the HR donor cassette, as shown in Figure S7D.

To integrate the reporters into the AAVS1 locus in U2OS cells, the reporter cassettes were subcloned into an AAVS1-targeting vector containing hygromycin-resistance gene. Integration was achieved using CRISPR/Cas9 with the AAVS1 gRNA (Table S1) and confirmed by PCR analysis. The EGFP-MMEJ reporter was also randomly integrated into the U2OS genome and clones with single integration was confirmed by ddPCR.

To generate the E14 mES (EGFP-MMEJ-ROSA) reporter cell line, the CAG-EGFP-MMEJ reporter, generated by replacing the CMV promoter of the CMV-EGFP-MMEJ reporter with the CAG promoter, was targeted to the ROSA locus by CRISPR/Cas9 (ROSA gRNA in Table S1). To generate Igh-MMEJ-For or Igh-MMEJ-Rev reporters, the CAG-EGFP-MMEJ cassette was cloned into a vector containing 1 kb homology arms to the Igh locus on both sides of the reporters in either forward or reverse directions. Igh-MMEJ-For or Igh-MMEJ-Rev reporters were targeted to the Igh locus in E14 mES cells by CRISPR/Cas9 (Igh gRNA in Table S1). The EBV/oriP-EGFP-MMEJ reporter was generated by inserting the CMV-EGFP-MMEJ reporter into the pCEP4 vector or the pCEP4-derived vector without the EBNA1 coding sequence.

Expression and purification of recombinant proteins

Coding sequence of the human Polθ helicase domain (1–894 aa) was amplified from pCDH-EF1-FHC-POLQ (Addgene, #64875) and inserted into the Sumo3 PolQM1 plasmid (Addgene, #78462, encoding Polθ-PolD) to replace the Polθ polymerase domain. The helicase-dead mutation K121A was introduced into Polθ helicase domain by QuikChange site-directed mutagenesis (Agilent, #200514). The Polθ polymerase domain (Polθ-PolD), helicase domain (Polθ-HelD) and RPA1/RPA2/RPA3 complex were expressed in E. coli Rosetta 2 (DE3) and purified as described^87,88.

GST-PCNA and GST-PCNA-Ub cloned into pGEX-6p-1 were expressed in E. coli Rosetta 2 (DE3)⁸⁹ and bound to glutathione resin (Clontech, 635607) in NETN buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40). PreScission cleavage was used to remove the GST tag on-beads in buffer (50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) at 4°C for 16 hours. The cleaved PCNA and PCNA-Ub in the supernatant were dialyzed into storage buffer (40 mM Tris-HCl, pH 8.0, 200 mM NaCl, 10% glycerol, 5 mM 2-mercaptoethanol).

In vitro assays of Polθ helicase and polymerase activities

Double-stranded DNA (dsDNA) substrates, with or without single-stranded DNA (ssDNA) overhangs, were generated by annealing of ssDNA oligos. Briefly, the indicated amount of ssDNA oligos were mixed in NEBuffer 2, heated in a water bath at 95°C for 5 minutes, and then allowed to cool slowly to room temperature.

DNA unwinding activity of Polθ-HelD was determined as described⁹⁰. 5 nM ³²P-5’-radiolabeled pre-annealed DNA substrate was mixed with Polθ-HelD WT or K121A mutant (20 nM) in reaction buffer (25 mM Hepes-NaOH, pH 7.0, 40 mM KCl, 5% glycerol, 1 mM MgCl₂, 2 mM DTT, 0.01% NP-40), followed by the addition of ATP (2 mM), cold ssDNA trap (200 nM) and RPA complex (20 nM) as indicated for 15 minutes at 37°C.

To assay for DNA unwinding and strand displacement by Polθ-HelD, 10 nM ³²P-5’-radiolabeled ssDNA was mixed with 5 nM pre-annealed dsDNA substrate, Polθ-HelD WT or K121A mutant (20 nM), and ATP (2 mM), and RPA complex (20 nM) were added to the reactions as indicated, followed by incubation at 37°C for 15 minutes. The reaction was stopped by addition of 10 mg/ml proteinase K (NEB, P8107), 10 mM EDTA, and 0.08% SDS at final concentration, and resolved on 12% non-denaturing polyacrylamide gels and visualized by phosphor imaging.

To access the activities of Polθ-HelD and Polθ-PolD in strand displacement DNA synthesis, the strand displacement reaction by Polθ-HelD was supplemented with 20 nM Polθ-PolD and 0.1 mM dNTPs and incubated for another 20 minutes at 37°C. The reactions were terminated by adding an equal volume of denaturing stop buffer (90% formamide, 50 mM EDTA), resolved on 10% denaturing polyacrylamide gels (8 M urea), and visualized by phosphor imaging.

To examine the ability of Polθ-PolD to extend DNA synthesis from 3’ unpaired bases, 100 nM ³²P-5’-radiolabeled DNA substrates were incubated with Polθ-PolD (20 nM) and 0.5 mM dATP in reaction buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl₂) for indicated time at 37°C. The reactions were terminated by adding equal volume of denaturing stop buffer (90% formamide, 50 mM EDTA) and resolved on 15% denaturing polyacrylamide gels (8 M urea) and visualized by phosphor imaging.

Pull-down experiments

To test interactions between the helicase or polymerase domain of Polθ and the RPA complex, GST-fused Polθ helicase (Polθ-HelD) and polymerase (Polθ-PolD) domains were expressed in E. coli Rosetta 2 (DE3). Bacteria pellets were lysed in NETN buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40) containing protease inhibitors, aprotinin (2 μg/ml) and PMSF (100 μg/ml) and incubated with Glutathione resin for 3 hours at 4°C, followed by three washes with NETN buffer. Purified RPA1/RPA2/RPA3 (RPA) complex was incubated with the glutathione resin bound with GST-fused Polθ-HelD and Polθ-PolD or GST in NETN buffer for 3 hours at 4°C. After extensive washing, GST pull-down samples were subjected to 15% SDS-PAGE, followed by Western blot analysis using anti-RPA1 and anti-RPA2 antibodies.

To examine interactions between PCNA and PIF1, 293T cells transfected with the pCDH-CMV-3×Flag-PIF1-neo plasmid were lysed in NETN buffer and incubated with glutathione resin bound to GST-PCNA or GST-PCNA-Ub for 3 hours at 4 °C. After extensive washing, the pull-down samples were analyzed by anti-Flag Western blotting. Inputs of GST, GST-PCNA, and GST-PCNA-Ub were visualized by Coomassie blue staining.

To examine interactions between PCNA and Polθ, 293T cells expressing Flag-Polθ were lysed in NETN buffer and incubated with purified PCNA or PCNA-Ub, which were cleaved from GST-PCNA or GST-PCNA-Ub using PreScission. GST alone was used as a control. Anti-Flag immunoprecipitation was performed using M2 antibody (Millipore), followed by Western blotting with anti-PCNA and anti-Flag antibodies. Inputs of purified GST, PCNA, and PCNA-Ub were visualized by Coomassie blue staining.

Generation of POLQ knock-out and mutants by CRISPR

A pair of POLQ KO gRNAs (POLQ KO gRNA1 and gRNA2 in Table S1) targeting mouse POLQ exon 1 were subcloned into a CRISPR vector expressing pSpCas9 and mCherry marker. Transfections were carried out in mES cells containing the EGFP-MMEJ reporter with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s recommendations. 72 hours after transfection, mCherry-positive cells were sorted by flow cytometry and single cells were placed into individual wells of a 96-well plate pre-coated with 0.1% gelatin. POLQ knock-out clones were screened by PCR amplification using a pair of primers (Mouse POLQ KO screening-F and Mouse POLQ KO screening-R in Table S2) and were confirmed by Sanger sequencing.

POLQ helicase and polymerase mutants in E14 cells were generated following the described strategy⁸⁷. For making the Polθ helicase mutant (K120G) in E14 cells harboring the EGFP-MMEJ reporter, a pair of gRNAs (K120G gRNA1 and gRNA2 in Table S1) were subcloned into the pX459 V2.0-Cas9^D10A vector, and the resulting gRNA plasmids were co-transfected together with a donor cassette using Lipofectamine 2000 (Invitrogen). The donor plasmid contains 690 bp and 565 bp homology arms on the upstream and downstream of the mutation K120G. Similarly, for Polθ polymerase mutant (D2494P/E2495R) in E14 cells, a pair of gRNAs (D2494P/E2495R gRNA1 and gRNA2 in Table S1) were subcloned in pX459 V2.0-Cas9^D10A and were co-transfected together with a donor template containing 700 bp and 544 bp homology arms on the upstream and downstream of the mutation D2494P/E2495R. 24 hours after transfection, cells were selected for puromycin (1 μg/ml, 2 days) to enrich transfected cells, and then maintained in regular media for 3–4 days. Single clones were expanded and screened for the knock-in mutations by genomic DNA PCR sequencing (Primers for K120G: K120G screening-F and K120G screening-R; Primers for D2494P/E2495R: D2494P/E2495R screening-F and D2494P/E2495R screening-R in Table S2).

EGFP-MMEJ, EGFP-MMEJ/mCherry-BIR and mCherry-MMEJ/EGFP-mCherry-HR reporter assays

For protein knockdown in U2OS cells carrying reporters, cells were infected with concentrated lentiviruses expressing shRNAs for indicated genes with the empty vector as a control. 24 hours after infection, cells were replaced with fresh media and subjected to blasticidin selection (10 μg/ml, 2 days). To perform reporter assays, U2OS cells were infected by lentiviruses, encoding the indicated gRNAs cloned in the lentiCRISPRv2 vector or its derived vectors, expressing either Cas9^D10A or Cas9^H840A, followed by puromycin selection (2 μg/ml, 2 days). EGFP or mCherry-positive events were analyzed 4 days after viral infection of Cas9 or Cas9n using a BD Accuri C6 flow cytometer. The gRNA cleavage sites in the EGFP-MMEJ, EGFP-MMEJ/mCherry-BIR, and mCherry-MMEJ/EGFP-mCherry-HR reporters are identical; all reporters use the same gRNAs for the repair assays.

To perform reporter assays in E14 cells, MMEJ gRNA2 and gRNA6 in Table S1, targeting the EGFP-MMEJ reporter, were cloned in the pSpCas9(BB)-2A-Puro (PX459) V2.0 vector (Addgene plasmid # 62988) or its derived vectors that express Cas9^D10A or Cas9^H840A, followed by transfection to E14 cells carrying the EGFP-MMEJ reporter at the ROSA or Igh locus. Transfection was carried out with a total of 3×10⁵ cells per well in a 24-well plate with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions (1 μg plasmid DNA per well). 4 hours later, transfection mixes were replaced with fresh medium, and after another 24 hours, 1 μg/ml puromycin was added for 48 hours to eliminate untransfected cells. Cells were analyzed for EGFP-positive events by flow cytometry 4 days post infection.

Droplet digital PCR (ddPCR) assay for quantification of double-strand breaks

To determine the percentage of cells with DSB formation at the site of gRNA cleavage, ddPCR was performed as described⁹¹. Two amplicons were designed, of which, one includes the gRNA/Cas9 target site, and the other is a proximal uncut site as a control. A dual-quenched probe was designed for each amplicon, which was labeled using FAM or HEX at the 5’ of the probe for target site and control site. 50 ng genomic DNA was used as template in a 20 μl reaction containing 10 μl 2×ddPCR Supermix for Probes (No dUTP) (Bio-Rad, 186–3023), 900 nM of each primers and 250 nM probes for both target and control sites. BamHI-HF (NEB, R3136) was added to the reaction at a ratio of 1:100 for better separation of signals. Droplets were generated using QX200 Droplet generator (Bio-Rad). The amplification was performed on a C1000 Touch Thermal Cycler with the following conditions: 95°C for 10 min, 40 cycles of (94°C for 30 s, 60°C for 30 s, 72°C for 1 min), 98°C for 10 min, 12°C hold. Droplets were scanned using the QX200 Droplet Digital PCR system (Bio-Rad). Droplets in each fluorescent channel (FAM/HEX) were plotted and distinguished in clusters using a global threshold to bin droplets to positive and negative labels. The DSB frequency was calculated as [target-, control+]/([target-, control+] + [target+, control+]).

Light-inducible CRISPR

Caged gRNA for EGFP-MMEJ reporter was designed as described⁴⁸, and synthesized by Bio-Synthesis Inc. 200 pmol caged gRNA was mixed with 200 pmol Alt-R^™ CRISPR-Cas9 tracrRNA (IDT, 1072532), heated to 95°C for 5 minutes, and allowed to cool to room temperature for 5 minutes. 3 μl of 10 μg/μl Alt-R^™ S.p. Cas9 D10A Nickase V3 (IDT, 1081062) was added to the annealed mixture of caged gRNA and tracrRNA, and was incubated at room temperature for 20 minutes to form RNP complex. 1×10⁷ U2OS (EGFP-MMEJ) cells were resuspended in 90 μl SE solution and supplied with 20 μl Supplement solution from SE Cell line 4D-NucleofectorTM X kit (Lonza, V4XC-1032). Electroporation was performed according to the manufacturer’s instructions on the 4D-Nucleofector Core Unit (Lonza) with code DN-100. Cells were plated in complete DMEM and incubated at 37°C for 16 hours for recovery.

Synchronization in G1 phase and cell cycle analysis of U2OS cells

To synchronize U2OS cells in G1 phase, Palbociclib was added to 70% confluent cells at a final concentration of 0.25 μM for 20 hours. Cells remaining in G1 arrest or released from G1 by replacing with fresh medium were trypsinized, and vigorously resuspended in 1 ml PBS, followed the dropwise addition of 4 ml cold ethanol. After fixation for 1 hour at 4°C, cells were stained with 10 μg/mL Propidium Iodide in the presence of 0.2 mg/mL RNase A. Cell cycle profile was analyzed by flow cytometry and the percentage of cells in different cell cycle phases was analyzed by FlowJo.

Analysis of Cas9^WT, Cas9^D10A or Cas9^H840A-induced indels at genomic loci by deep sequencing

gRNAs targeting human LBR locus (LBRg1 and LBRg2 in Table S1) and mouse Igh locus (Igh-g2 in Table S1) were cloned into pSpCas9(BB)-2A-Puro (PX459) V2.0 vector (Addgene plasmid # 62988) and LentiCRISPR v2 vector (Addgene plasmid # 52961) or their derived vectors expressing Cas9^D10A or Cas9^H840A. 72 hours after transfection of plasmids encoding gRNAs with Cas9^WT, Cas9^D10A or Cas9^H840A, genomic DNA was extracted using DNeasy Blood & Tissue Kits (QIAGEN, 69504). Libraries of repair products were generated by PCR amplification using primers for LBR and Igh locus listed in Table S2, producing ~250 bp DNA fragments surrounding the gRNA target sites. A second round of PCR was performed with P5 and P7 primers containing i5 and i7 index sequences, and subjected to GENEWIZ for paired-end 150 bp deep sequencing on illumina NovaSeq platform. Quality control of raw reads was performed using FastQC. Paired-end reads were merged using FLASH, followed by trimming and demultiplexing with Cutadapt. High-quality reads were aligned to the reference genome using BBMap. Repair patterns were analyzed based on CIGAR values using Python. Data visualization and statistical analyses were conducted in R and GraphPad Prism.

DSB end capture

To directly demonstrate DSBs formation at the EGFP-MMEJ reporter after Cas9 and Cas9n cleavage, we adapted DSBcapture⁴⁷ for DSB end capture at a specific genomic locus (Fig. S4A). 24 hours after infection by lentiviruses expressing gRNA and Cas9^WT or Cas9^D10A, 1×10⁷ cells were fixed with 2% formaldehyde at room temperature for 30 minutes, followed by addition of glycine to a final concentration of 125 mM to quench formaldehyde. Cells were collected, washed with ice cold PBS and lysed in lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2% NP-40, 1 mM DTT, cOmplete proteinase inhibitors) by gently rotating at 4°C for 90 minutes. After centrifugation at 1200 rpm for 5 minutes, nuclei were resuspended in nucleus break buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.3% SDS, 1 mM DTT) and incubated at 37°C for 45 minutes. Nuclei pellets were then resuspended in NEBuffer 2 (10 mM Tris-HCl, pH8.0, 50 mM NaCl, 10 mM MgCl₂, 1 mM DTT) with 0.1% Triton X-100, and proteinase K was added to a final concentration of 100 μg/mL. After incubation at 37°C for 8 minutes, an equal volume of NEBuffer 2 containing 0.1% Triton X-100 and 2 mM PMSF was added to inactivate proteinase K. Nuclei were then washed twice by resuspending in NEBuffer 2 with 0.1% Triton X-100 followed by centrifugation at 1200g for 10 minutes. Nuclei were washed with Blunting Buffer (100 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl₂, 0.025 % Triton X-100, 5 mM DTT) with 100 μg/mL BSA and were blunt-ended by the addition of 2 μL T4 DNA polymerase (NEB, M0203), 0.5 μL T4 PNK (NEB, M0201) and 4 μL 2.5mM dNTPs in a 50 μl volume, followed by incubation at 25°C for 45 minutes. Nuclei were then washed three times with NEBuffer 2 with 0.1% Triton X-100, resuspended in NEBuffer 2 and A-tailed using 3 μL Klenow Fragment 3’−5’ exo- (NEB, M0212L) and 1 μL 5 mM dATP in a final volume of 50 μL at 37°C for 45 minutes. Following A-tailing, nuclei were washed three times with NEBuffer 2 with 0.1 % Triton X-100, once with T4 DNA ligase reaction buffer containing 0.1% Triton X-100 at 4°C and resuspended in T4 DNA ligase reaction buffer. Annealed adaptor containing UMI (UMI-For and UMI-Rev in Table S2), was then ligated to DNA ends using T4 DNA ligase (NEB, M0202M) for 15 hours at 16°C in a final reaction volume of 50 μL. For genomic DNA extraction, the ligation mixture was treated with proteinase K (200 μg/ml) for 30 minutes at 55°C, followed by incubation at 65°C for 30 minutes, and DNA was precipitated using isopropanol. The pelleted DNA was resuspended in 50 μL of water. The DSB capture library was then amplified by nested PCR, followed by an additional PCR to add illumina i5 and i7 sequences to the ends of the amplicons. The final PCR products were subjected to paired-end 150 bp deep sequencing on the illumina NovaSeq platform. The PCR primers for DSB end capture after Cas9 and Cas9n cleavage of the EGFP-MMEJ reporter are listed in Table S2.

Lentiviral infection for protein expression and shRNA interference

The coding sequences of PCNA, Flag-RPA2, RPA2-FKBP12^F36V were subcloned into the pCDH-CMV-EF1-Puro vector (NovoPro, V006738). RPA2 S4A/S8A/T21A/S33A and S4D/S8D/T21D/S33D mutants, as well as the PCNA-K164R mutant, were generated using QuikChange site-directed mutagenesis kit (Agilent, 200514). Short hairpin RNAs (shRNAs) targeting the indicated genes were cloned into the pLKO.1-blast vector (Addgene #26655). shRNA target sequences were listed in Table S4.

For lentivirus production, 293T cells were co-transfected with lentiviral expression plasmids, along with pMD2.G (Addgene, #12259) and psPAX2 (Addgene, #12260) using standard calcium chloride protocol. Lentivirus particles released into the cell culture medium were collected 72 hours after 293T transfection. The cell culture medium was filtered and concentrated using PEG-800 (40% W/V) containing NaCl (1.2 M). After lentiviral infection, cells were selected with blasticidin (10 μg/ml, 2 days) or puromycin (2 μg/ml, 2 days). Protein expression levels of the targeted genes were verified by Western blot analysis.

Cell viability assay

Cells were seeded in 96-well plates at a density of 2×10³ cells per well and treated with indicated concentration of drugs for 72 hours. Subsequently, 100 μl of cell medium from each well was mixed with 20 μl Cell Counting Kit-8 (Dojindo, CK04) and incubated at 37°C for 2 hours. Relative viability was determined by measuring the emission at 490 nm using 800TS Microplate Reader (BioTek), and normalized to control cells treated with DMSO.

Immunoblotting

Cells from a confluent 6 cm plate were lysed in NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) at 4°C for 30 minutes. For Polθ, cell pellets were lysed in 2% SDS, 125 mM Tris-HCl (pH 6.8) supplemented with protease and phosphatase inhibitor cocktail on ice, as described⁹². Cell debris was removed by centrifugation. After adding 2×loading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 200 mM DTT) to the cell lysates, the samples were heated at 95°C for 5 minutes and separated on SDS-PAGE. Antibodies used in immunoblotting are listed in Key resources table.

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from cell lines using TRIzol reagent (Invitrogen) according to manufacturer’s instructions. cDNA was synthesized from 1 μg of total RNA as the template by reverse transcription using the iScript cDNA Synthesis Kit (Bio-Rad, 1708890) according to the manufacturer’s protocol. The synthesized cDNA was quantified by qPCR using Luna Universal qPCR Master Mix (NEB, M3003) in a C1000 Thermal Cycler (Bio-Rad).

DNA fiber assay

Cells were first pulse labeled with 40 μM CldU (Sigma-Aldrich, C6891) for 30 minutes, followed by treatment with 2 mM HU for 2 hours, and then labeled with 200 μM IdU (Sigma-Aldrich, I7125) for 30 minutes. After trypsinization, cells were resuspended in PBS and then labeled and unlabeled cells were mixed in a ratio of 1:6.5 μl of cell suspension was placed on a glass slide, then mixed with 7.5 μl lysis solution (200 mM Tris-HCl, pH 7.5, 50 mM EDTA, 0.5% SDS) for 3 minutes. Tilt the slide to a suitable angle to spread the DNA. The fibers were fixed in a 3:1 (vol/vol) methanol: acetic acid solution. DNA was denatured with HCl and blocked with 5% bovine serum albumin (BSA) in PBS for 1 hour after washing. CldU and IdU detection were performed using rat anti-BrdU (Abcam, ab6326) and mouse anti-BrdU (BD Biosciences, 347580) primary antibodies for 2 hours at 37°C, followed by incubation with Alexa 594 anti-rat (Invitrogen, A11007) and Alexa 488 anti-mouse (Invitrogen, A11029) for 1 hour at room temperature. Coverslips were washed with PBS with 0.1% Tween-20 and mounted with Prolong Gold antifade reagent (Invitrogen, P10144). DNA fibers were imaged with a LSM 780 confocal laser scanning microscope and analyzed using ImageJ software (NIH, USA). At least 100 replication forks were analyzed per experimental condition, with the results representing the mean of three independent experiments.

In situ Proximity Ligation Assay (PLA)

Proximity ligation assay was performed following the manufacturer’s recommendation (Duolink, Sigma-Aldrich, DUO92101). Cells treated with HU (2 mM) for 24 hours were fixed in 2% paraformaldehyde for 20 minutes. Fixed cells were treated with 0.5% Triton X-100 for 10 minutes for permeabilization. Cells were then blocked with 3% BSA for 30 minutes at 37°C in a humidity chamber, followed by incubation with primary antibodies diluted in Duolink Antibody Diluent overnight at 4°C. Coverslips were fixed onto glass slides using ProLong Gold antifade mountant (Invitrogen, P10144) with DAPI. The PLA signals were visualized as distinct fluorescence spots, and images were captured with an Olympus confocal microscope using a 60X objective. The number of PLA signals was quantified using CellProfiler 4.2.6 software.

Chromatin immunoprecipitation (ChIP) assay

The recruitment of Flag-Polθ and γH2AX to the Cas9 cleavage site on the EGFP-MMEJ reporter was performed by ChIP assay. U2OS (EGFP-MMEJ) cells expression Flag-Polθ or RPA2-FKBP12^F36V was infected with lentivirus expressing gRNA2/Cas9^WT or gRNA2/Cas9^D10A. 24 hours after removal of the virus, cells were fixed with 1% formaldehyde for 10 minutes at room temperature. Glycine was added to the final concentration of 125 mM for 15 minutes at room temperature. After washing twice with cold PBS, cells were lysed and subjected to sonication. The supernatant was collected and incubated with Protein G dynabeads (Invitrogen, 10003D) which have been pre-loaded with indicated anti-Flag antibody or anti-γH2AX antibody for 4 hours at 4 °C, followed by washing with 1 ml TSE I (20 mM Tris-HCl, pH 8.1, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), TSE II (20 mM Tris-HCl, pH 8.1, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), buffer III (10 mM Tris-HCl, pH 8.1, 0.25 M LiCl, 1 mM EDTA, 1% NP-40, 1% deoxycholate), and TE. The protein-DNA complex was eluted from beads by elution buffer (1% SDS, 0.1M NaHCO₃), and cross-linking was reversed by adding in 4 μl of 5 M NaCl and incubating at 65 °C for 6 hours, followed by proteinase K digestion (NEB, P8107) for 2 hours at 42°C. DNA was recovered by column purification, and analyzed by RT-qPCR using primers MMEJ-ChIP-F and MMEJ-ChIP-R.

Immunofluorescence

The procedure for visualizing single-stranded DNA was described previously⁹³. In short, cells were cultured in 10 μM BrdU (Sigma-Aldrich, B5002) for 24 hours. After PBS washing, cells were incubated in pre-extraction buffer (10 mM PIPES, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, and 0.2% Triton X-100) for 5 minutes, fixed with 3% paraformaldehyde in PBS for 20 minutes. Subsequently, fixed cells were washed with PBS, treated with cold acetone for 30 seconds, washed again with PBS, and blocked in 2% BSA for 1 hour. Then blocked cells were incubated with the primary antibody at 4°C overnight. After washing three times with blocking buffer, the cells were incubated with appropriate secondary antibodies for 1 hour in the dark, then incubated in DAPI solution for 2 minutes. Cells were subjected to immunofluorescence analyses using an Olympus IX-81 fluorescence microscope.

Nuclear extract-based assay to analyze RPA2 phosphorylation

Nuclear extracts were prepared from U2OS cells as previously described⁶⁹. Nuclear extracts were incubated with 1 mM Mirin at 37°C for 10 minutes in reaction buffer containing 10 mM Hepes-KOH, pH 7.9, 50 mM KCl, 0.5 mM DTT, 4 μM Okadaic acid, 1 mM ATP, 0.1 mM MgCl2, 5 mM creatine phosphate and 1×Halt^™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, 78440). 1 μM 3’-biotinylated ssDNA was annealed to its complementary strand at a ratio of 1:1 to generate dsDNA substrate with 3’ overhang of indicated length. 200 nM annealed substrate was incubated with 1.5-fold streptavidin (Invitrogen, 434301) in reaction buffer at 25°C for 30 minutes. The reaction was initiated by mixing equal volume of DNA substrates with nuclear extracts, and incubated at 37°C for 30 minutes. The reaction was stopped with 2×SDS loading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 200 mM DTT) and resolved on 12% SDS-PAGE. RPA2 phosphorylation was determined by immunoblotting using phospho-RPA32 (S33) antibody (Bethyl, BLR363N). DNA sequences are listed in Table S2.

Replication timing analysis by Repli-seq

Repli-seq was performed as previously described⁹⁴. Briefly, cells were labeled with 100 μM BrdU (Sigma-Aldrich, B5002) for 2 hours and then fixed in 75% ethanol. Fixed cells were then sorted by FACS into early-S and late-S fractions based on propidium iodide staining of DNA. DNA was then purified from each fraction, sheared and adaptor-ligated with NEBNext Ultra II FS Library DNA Prep Kit for Illumina (NEB, E7805). BrdU-labeled nascent DNA library fragments were then enriched by immunoprecipitation with anti-BrdU antibody (BD, 555627). IP products are then PCR amplified and indexed with NEBNext Multiplex Oligos for Illumina (NEB, E6448). The libraries were sequenced on NovaSeq X Plus for 150 bp paired-end run to obtain at least 10 million clusters per library. The sequencing data were analyzed as previously described (https://github.com/ClaireMarchal/repli-seq)⁹⁴. Briefly, raw sequencing reads were mapped to reference genome using bowtie2, then converted to bam files using samtools. Following duplicated reads were removed using samtools, bam files were converted to bed files using 50kb genome windows. After reads number per each 50 kb window were obtained from “Early S fraction” and “Late S fraction” samples, replication timing which is log2[(reads in Early S fraction)/(reads in Late S fraction)] was calculated. This raw replication timing was scaled and smoothed using the method described in (https://github.com/ClaireMarchal/ScalingSmoothing). Scaling was performed using the scale function in R, followed by LOESS smoothing with a 300 kb window in R.

Quantification and statistical analysis

Statistical analysis was performed using GraphPad Prism 10. The data are presented as mean values ± SD from at least three independent experiments. Significant differences were determined by unpaired Student’s t-test between the two groups or two-way ANOVA test between multiple groups. The p value is labeled as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns (not significant) p > 0.05. The statistical significance of the replication timing difference was analyzed using SwitchRT (https://github.com/GilbertLab-dnadave/SwitchRT).

Supplementary Material

NIHMS2151773-supplement-1.pdf^{(3.2MB, pdf)}

Supplemental information

Document S1. Figures S1-S7, Table S1-S4, and supplemental references

Highlights.

Polθ-mediated MMEJ directly repairs seDSBs on broken forks (fork-MMEJ).
Fork-MMEJ is resection-independent, produces asymmetric deletions and requires RPA.
ATR controls the switch from fork-MMEJ to BIR coupled with the end resection extent.
Combined inhibition of ATR and Polθ synergistically kills cancer cells.

Acknowledgments

Sumo3 PolQM1 (Addgene # 78462) and pCDH-EF1-FHC-POLQ (Addgene # 64875) are gifts from Dr. Richard D. Wood (The University of Texas MD). pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene # 62988) and lentiCRISPR v2 (Addgene # 52961) are gifts from Dr. Feng Zhang (MIT). pLKO.1-blast (Addg ene # 26655) is a gift from Dr. Keith Mostov (UCSF). pCDH-CMV (Addgene # 72265) is a gift from Dr. Kazuhiro Oka (Baylor College of Medicine). pCW-Cas9 (Addgene # 50661) is a gift from Dr. Eric Lander & Dr. David Sabatini (MIT). Tet-On 3×Flag Polθ is a gift from Dr. Dale A. Ramsden (University of North Carolina, Chapel Hill). We thank Dr. Peiqing Sun (Wake Forest University) for the BJ and BJ (EMR) cells. This work was supported by research grants from NIH CA187052, CA197995, GM080677 and CA294646 (X.W.); NIH CA270335 (D.M.G.); the intramural program of the Center for Cancer Research, National Cancer Institute (ZIA BC010411 to M.I.A.) and National Natural Science Foundation of China (32270579 to S.L.).

Footnotes

Declaration of interests

The authors declare no competing interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS2151773-supplement-1.pdf^{(3.2MB, pdf)}

Data Availability Statement

Raw sequencing data (site-specific sequencing, nascent strand sequencing, Repli-seq) are available in the NIH Sequence Read Archive (SRA) as BioProject: PRJNA1235770. The original uncropped images are deposited at Mendeley Data: 10.17632/d79jttdxyc.4. The accession numbers of the published nascent strand sequencing of mouse embryonic stem cells are indicated in the Key Resources Table.
This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Key Resources Table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
mouse Anti- BrdU	BD Biosciences	Cat# 555627, RRID: AB_395993
rat anti-BrdU	Abcam	Cat# ab6326
mouse anti-BrdU	BD Biosciences	Cat# 347580, RRID: AB_400326
Rabbit anti-MRE11	Cell Signaling Technology	Cat# 4895
Rabbit anti-CtIP	Proteintech	Cat# 12624–1-AP, RRID: AB_2175250
Mouse anti-RPA1	Sigma-Aldrich	Cat# NA13
Rabbit anti-RPA2	Bethyl	Cat# A300–244A
Rabbit anti-PCNA	Cell Signaling Technology	Cat# 13110
Rabbit anti-MCM2	Proteintech	Cat# 10513–1-AP, RRID: AB_2142131
Rabbit anti-DNA2	Proteintech	Cat# 18727–1-AP, RRID: AB_10637998
Mouse anti-KU70	Santa Cruz Biotechnology	Cat# sc-17789
Mouse anti-HA	Santa Cruz Biotechnology	Cat# sc-7392
Mouse anti-Flag	Sigma-Aldrich	Cat# F1804
Rabbit anti-BLM	Bethyl	Cat# A300–110A
Rabbit anti-EXO1	Bethyl	Cat# A302–640A
Mouse anti-ATR	Santa Cruz Biotechnology	Cat# sc-515173
Rabbit anti-LIG3	Proteintech	Cat# 26583–1-AP, RRID: AB_2880562
Rabbit anti-β-Actin	Proteintech	Cat# 20536–1-AP, RRID: AB_10700003
Rabbit anti-γH2AX	Sigma-Aldrich	Cat# 07–164
Rabbit anti-Polθ	Cell Signaling Technology	Cat# 64708
Rabbit anti-PIF1	Affinity Bioscience	Cat# DF9256
Peroxidase AffiniPure Goat Anti-Mouse IgG(H+L)	Jackson ImmunoResearch Labs	Cat# 115–035-146, RRID: AB_2307392
Peroxidase AffiniPure Goat Anti-Rabbit IgG(H+L)	Jackson ImmunoResearch Labs	Cat# 111–035-144, RRID: AB_2307391
Alexa 594 Goat anti-rat IgG(H+L)	Invitrogen	Cat# A11007
Alexa 488 Goat anti-mouse IgG(H+L)	Invitrogen	Cat# A11029
Rabbit anti-Phospho RPA32 (S33)	Bethyl	Cat# BLR363N
Bacterial and virus strains
E. coli Rosetta 2 (DE3)	Novagen	Cat# 71397
Chemicals, peptides, and recombinant proteins
dTAG-13	MCE	Cat# HY-114421
BrdU	Sigma-Aldrich	Cat# B5002
1×Halt^™ Protease and Phosphatase Inhibitor	Thermo Scientific	Cat# 78440
streptavidin	Invitrogen	Cat# 434301
proteinase K	NEB	Cat# P8107
CldU	Sigma-Aldrich	Cat# C6891
IdU	Sigma-Aldrich	Cat# I7125
Doxycycline	MCE	Cat# HY-N0565
Mirin	MCE	Cat# HY-19959
ART558	MCE	Cat# HY-141520
VE822	MCE	Cat# HY-13902
Palbociclib	MCE	Cat# HY-50767
cOmplete proteinase inhibitors	Roche	Cat# 11697498001
T4 DNA polymerase	NEB	Cat# M0203
T4 PNK	NEB	Cat# M0201
T4 DNA ligase	NEB	Cat# M0202
Alt-R^™ S.p. Cas9 D10A Nickase V3	IDT	Cat# 1081062
PreScission protease (HRV3C) recombinant protein	Abnova	Cat# P7352
ProLong Gold antifade mountant	Invitrogen	Cat# P10144
Critical commercial assays
NEBNext® Ultra^™ II FS DNA Library Prep Kit for Illumina	NEB	Cat# E7805
QuikChange site-directed mutagenesis kit	Agilent	Cat# 200514
Glutathione sepharose resin	Clontech	Cat# 635607
Cell Counting Kit-8 (CCK-8)	Dojindo	Cat# CK04
Protein G dynabeads	Invitrogen	Cat# 10003D
iScript cDNA Synthesis Kit	Bio-Rad	Cat# 1708890
Luna Universal qPCR Master Mix	NEB	Cat# M3003
Duolink® In Situ Detection Reagents Red	Sigma-Aldrich	Cat# DUO92008
Duolink® In Situ Detection Reagents Green	Sigma-Aldrich	Cat# DUO92014
DNeasy Blood & Tissue Kits	QIAGEN	Cat# 69504
SE Cell line 4D-NucleofectorTM X kit	Lonza	Cat# V4XC-1032
ddPCRTM Supermix for Probes (No dUTP)	Bio-Rad	Cat# 186–3023
Lipofectamine® 2000 Transfection Reagent	Invitrogen	Cat# 11668019
NEBNext® Multiplex Oligos for Illumina	NEB	Cat# E6448
Deposited data
Raw data of site-specific PCR deep sequencing, Nascent strand sequencing of U2OS cells, Repli-seq data of mouse Embryonic stem cells	This paper	NIH Sequence Read Archive: PRJNA1235770
Original images of uncropped gels	This paper	Mendeley Data: 10.17632/d79jttdxyc.4
Nascent strand sequencing of mouse Embryonic stem cells	⁵⁷	GEO: GSM3814167, GSM3814168, GSE131699
Experimental models: Cell lines
Human: 293T	ATCC	CRL-3216
Human: U2OS	ATCC	HTB-96
Human: T98G	ATCC	CRL-1690
Human: RPE1(hTERT RPE-1)	ATCC	CRL-4000
Human: BJ	ATCC	CRL-2522
E14(ES-E14TG2a)	ATCC	CRL-1821
Oligonucleotides
Alt-R^™ CRISPR-Cas9 tracrRNA	IDT	Cat# 1072532
Oligos used as DNA substrate and PCR primers	Table S2	N/A
Recombinant DNA
EGFP-MMEJ-AAVS1 plasmid	This paper	N/A
EGFP-MMEJ/mCherry-BIR plasmid	This paper	N/A
mCherry-MMEJ/EGFP-mCherry-HR plasmid	This paper	N/A
pCDH-CMV	Addgene	# 72265
pCDH-CMV-ISceI-Puro	⁸⁷	N/A
pCDH-CMV-Flag-RPA2-Neo	This paper	N/A
pCDH-CMV-Flag-RPA2(S4A/S8A/T21A/S33A)-Neo	This paper	N/A
pCDH-CMV-Flag-RPA2(S4D/S8D/T21D/S33D)-Neo	This paper	N/A
pCDH-CMV-Flag-PIF1-Neo	This paper	N/A
pCDH-CMV-HA-RPA2-FKBP12^F36V	This paper	N/A
pCDNA-HA-Polθ-Neo	This paper	N/A
Tet-On 3×Flag Polθ	Dr. Dale A. Ramsden	N/A
pCEP4	NovoPro	Cat# V012686
EBV/oriP-EGFP-MMEJ plasmid	This paper	N/A
EBV/oriP-EGFP-MMEJ plasmid (no EBNA1)	This paper	N/A
pCW-Cas9	Addgene	# 50661
pCW-Cas9^D10A	This paper	N/A
pLKO.1-blast	Addgene	# 26655
pMD2.G	Addgene	# 12259
psPAX2	Addgene	# 12260
pSpCas9(BB)-2A-Puro (PX459) V2.0	Addgene	# 62988
lentiCRISPR v2	Addgene	# 52961
lentiCRISPR v2-D10A	This paper	N/A
lentiCRISPR v2-H840A	This paper	N/A
pCDH-EF1-FHC-POLQ	Addgene	# 64875
Sumo3 PolQM1	Addgene	# 78462
pSumo3-Polθ-HelD	This paper	N/A
pSumo3-Polθ-HelD(K121A)	This paper	N/A
pET28a-RPA1/RPA2/RPA3	This paper	N/A
Software and algorithms
ImageJ	⁸⁴	https://imagej.net/ij/
GraphPad Prism10	GraphPad	https://www.graphpad.com/
CellProfiler 4.2.6	CellProfiler	https://cellprofiler.org/
FlowJo v10	BD	https://www.flowjo.com/

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PERMALINK

Microhomology-mediated end joining acts directly on replication forks to repair single-ended double strand breaks

Shibo Li

Yuqin Zhao

Youhang Li

Sameer Bikram Shah

Yanmeng Shi

Tran Nguyen

Zi Wang

Chia-Yu Chang

Anagh Ray

Te-Hsuan Bu

Salvatore Loguercio

Takayo Sasaki

Jonathan H Sussman

Hailong Wang

David M Gilbert

Mirit I Aladjem

Xiaohua Wu

Summary

eTOC blurb

Graphical Abstract

Introduction

Results

MMEJ is induced upon fork breakage after Cas9n cleavage.

Figure 1. MMEJ is induced at broken forks by Cas9 nickase (Cas9n).

MMEJ at broken forks shows asymmetric features.

MMEJ induced after Cas9 and Cas9n exhibits different genetic dependence.

Figure 2. Distinct genetic requirements of MMEJ induced by Cas9 and Cas9n.

Figure 7. ATR, activated by end resection, specifically inhibits fork-MMEJ.

Fork-MMEJ is strongly suppressed by NH tails of 2 nucleotides (nt) or longer 3’ to the MH.

Figure 3. Fork-MMEJ is suppressed by ≥ 2 nt NH tails at the 3’ ends of parental broken strands.

Fork-MMEJ is used more frequently at seDSBs on the leading strands induced by Cas9n.

Figure 4. MMEJ preferentially repairs Cas9n-induced seDSBs on the leading strands.

Fork-MMEJ functions together with BIR to repair seDSBs

Figure 5. Fork-MMEJ functions together with BIR to repair seDSBs at broken forks.

Figure 6. Both MMEJ and BIR are important for replication restart.

ATR suppresses fork-MMEJ and shifts repair toward BIR in conjunction with end resection.

Inhibition of Polθ and ATR shows a synergistic effect to eradicate cancer cells under replication stress

Discussion

The mechanism underlying fork-MMEJ differs from cMMEJ at deDSBs.

A proposed working model of fork-MMEJ for seDSB repair.

Fork-MMEJ directly acts on seDSBs, alongside BIR, to repair replication-associated fork breakage.

ATR drives the transition of fork-MMEJ to BIR.

Limitations of the study

Resource availability

Lead contact

Materials availability

Data and code availability

STAR Methods

Experimental model and study participant details

Cell lines and cell culture

Method details

Plasmid construction and generation of the reporter cell lines

Expression and purification of recombinant proteins

In vitro assays of Polθ helicase and polymerase activities

Pull-down experiments

Generation of POLQ knock-out and mutants by CRISPR

EGFP-MMEJ, EGFP-MMEJ/mCherry-BIR and mCherry-MMEJ/EGFP-mCherry-HR reporter assays

Droplet digital PCR (ddPCR) assay for quantification of double-strand breaks

Light-inducible CRISPR

Synchronization in G1 phase and cell cycle analysis of U2OS cells

Analysis of Cas9WT, Cas9D10A or Cas9H840A-induced indels at genomic loci by deep sequencing

DSB end capture

Lentiviral infection for protein expression and shRNA interference

Cell viability assay

Immunoblotting

Reverse transcription-quantitative PCR (RT-qPCR)

DNA fiber assay

In situ Proximity Ligation Assay (PLA)

Chromatin immunoprecipitation (ChIP) assay

Immunofluorescence

Nuclear extract-based assay to analyze RPA2 phosphorylation

Replication timing analysis by Repli-seq

Quantification and statistical analysis

Supplementary Material

Highlights.

Acknowledgments

Footnotes

References

Analysis of Cas9^WT, Cas9^D10A or Cas9^H840A-induced indels at genomic loci by deep sequencing