53BP1-the ‘Pandora’s box’ of genome integrity

Abstract

53BP1 has several functions in the maintenance of genome integrity. It functions as a key mediator involved in double-strand break (DSB) repair, which functions to maintain a balance in the repair pathway choices and in preserving genomic stability. While its DSB repair functions are relatively well-characterized, its role in DNA replication and replication fork protection is less understood. In response to replication stress, 53BP1 contributes to fork protection by regulating fork reversal and restart. It helps maintain replication fork stability and speed, with 53BP1 loss leading to defective fork progression and increased sensitivity to replication stress agents. However, 53BP1’s precise role in fork protection remains debated, as some studies have not observed protective effects. Therefore, it is critical to determine the role of 53BP1 in replication to better understand when it promotes replication fork protection, and the underlying mechanisms involved. Moreover, 53BP1’s function in replication stress extends beyond its activity at active replication forks; it also forms specialized nuclear bodies (NBs) which protect stretches of under-replicated DNA (UR-DNA) transmitted from a previous cell cycle to daughter cells through mitosis. The mechanism of 53BP1 NBs in the coordination of replication and repair events at UR-DNA loci is not fully understood and warrants further investigation. The present review article focuses on elucidating 53BP1’s functions in replication stress (RS), its role in replication fork protection, and the significance of 53BP1 NBs in this context to provide a more comprehensive understanding of its less well-established role in DNA replication.

1. Introduction

The faithful replication of genetic material during cell division is fundamental to maintaining genome stability. However, DNA is perpetually exposed to endogenous and exogenous damaging agents that threaten this stability. Among various types of DNA damage, double-strand breaks (DSBs) are particularly hazardous, as even a single unrepaired DSB can induce cell senescence or death [1, 2]. Moreover, DSBs can lead to chromosomal instability manifested as amplifications, deletions, and translocations—processes intimately linked to tumorigenesis [3]. To combat these threats, cells have evolved a sophisticated arsenal of proteins dedicated to DSB repair. One such pivotal protein is the p53-binding protein 1 (53BP1), initially identified as an interactor of the tumor suppressor p53 [4]. 53BP1 has emerged as a multifaceted guardian of genome stability [5], with roles spanning several critical cellular processes:

1. Regulation of DNA Repair Pathway Choice: 53BP1 promotes non-homologous end-joining (NHEJ) by counteracting BRCA1-mediated end resection and homologous recombination (HR), particularly in the G1 phase of the cell cycle.

2. Scaffolding for Repair Factors: It serves as a platform for the assembly of DNA damage repair signaling complexes.

3. ATM Signaling Mediation: 53BP1 facilitates ATM-dependent signaling in response to DSBs, amplifying the DNA damage response.

4. Telomere Protection: It promotes NHEJ at deprotected telomeres, preventing detrimental chromosome fusions.

5. Immunological Functions: 53BP1 is crucial for class-switch recombination (CSR) and long-range V(D)J recombination, processes vital for adaptive immunity.

The functional versatility of 53BP1 is underpinned by its modular structure, comprising several domains, each contributing to specific aspects of its function. These domains include:

• Oligomerization Domain (OD)

• Tudor Domains

• Ubiquitin-Dependent Recruitment (UDR) motif

• Nuclear Localization Signal (NLS)

• BRCT Domains

Each domain plays a distinct role in 53BP1’s recruitment, activation, and interaction with other repair factors, collectively enabling its diverse functions in maintaining genome stability. The minimal focus forming region (FFR) is crucial for 53BP1 recruitment to DSBs, binding both H4K20me2 and H2AK15Ub histone marks via its tandem Tudor and UDR domains, respectively [6]. 53BP1 accumulation at DSB sites requires both histone marks, with H2AK15 ubiquitination dependent on RNF8 and RNF168 E3 ligase activities [7–9]. 53BP1 associates with TIRR (Tudor Interacting Repair Regulator, also known as NUDT16L1, or Syndesmos) via its tandem Tudor domain, inhibiting 53BP1 binding to H4K20me2 [10, 11]. Upon DNA damage, this complex dissociates, allowing 53BP1 to bind H4K20me2 near DSBs [12–14]. The OD and LC8 domain are essential for 53BP1 accumulation at DNA damage sites [15] and binds its constitutive partner DYNLL1, critical for anti-resection at DNA ends [16–18]. The N-terminus S/TQ sites interact with PTIP and RIF1, recruiting Shieldin, CST, and Polα/Primase to promote efficient NHEJ [19]. The C-terminus, including the BRCT domain, is involved in heterochromatin DSB repair and p53 transactivation [20–24]. The intricate involvement of 53BP1 in multiple facets of DNA repair and genome maintenance underscores its significance in cellular homeostasis and disease prevention. Understanding the mechanisms by which 53BP1 operates not only provides insights into fundamental cellular processes but also offers potential avenues for therapeutic interventions in cancer and other genomic instability-related disorders.

In addition to 53BP1’s role in controlling end resection and DSB repair pathway choice, 53BP1 has also been characterized in the context of replication, where it localizes to replication forks in response to replication stress [25, 26]. Several studies have reported that 53BP1 exhibits protective functions at replication forks, demonstrating that the loss of 53BP1 leads to reduced replication fork speed, increased fork reversal, and heightened sensitivity of cells to replication stress agents [27–29]. However, other studies have found no evidence supporting a protective role for 53BP1 at replication forks [30, 31]. These conflicting observations can be partly attributed to differences in cell lines, experimental methodology and the specific cellular contexts in which these studies were conducted [32]. Therefore, it is crucial to carefully consider the existing literature on 53BP1 and replication stress to better understand when it promotes replication fork protection, and the underlying mechanisms involved. Furthermore, 53BP1’s function in replication stress extends beyond its activity at active replication forks; it also forms specialized nuclear bodies (NBs), distinct cellular structures separate from DNA damage foci [33]. These NBs shield stretches of under-replicated DNA (UR-DNA) transmitted from a previous cell cycle to daughter cells through mitosis [33, 34]. The mechanism of 53BP1 NBs in the coordination of replication and repair events at UR-DNA loci is not fully understood and warrants further investigation [33, 35]. In this review, we focus on elucidating 53BP1’s functions in replication stress (RS), its role in replication fork protection, and the significance of 53BP1 NBs in this context to provide a more comprehensive understanding of its less well-established role in DNA replication.

2. 53BP1 Nuclear Bodies (NBs) in the maintenance of genome stability

The genome’s dynamic organizational properties create an intricate nuclear environment. Genome maintenance occurs through multiple mechanisms: the spatial and temporal organization of nuclear processes, chromatin arrangement into higher-order domains, and the specialized positioning of chromosomes within the nucleus [36]. Within organized chromosome territories, functionally distinct NBs concentrate various nuclear factors, including transcription factors, RNAs, and proteins [37–39]. Examples of NBs include histone locus bodies for histone mRNA processing, Cajal bodies involved in RNA metabolism, and paraspeckles associated with specific stress responses and RNA-protein interactions [37, 38]. NBs rapidly exchange components with the surrounding nucleoplasm, reflecting changes in gene expression and genomic stability [40]. 53BP1 NBs form in response to specific cellular stresses during the G1 phase of the cell cycle [34, 41]. Initially described as Oct1/PTF/transcription (OPT) domains, these structures form around DNA damage sites generated during mitosis at under-replicated genomic loci [34, 42]. When cells enter mitosis with under-replicated DNA (UR-DNA), resulting DNA lesions are transmitted to daughter cells and condensed into large, 53BP1-dependent compact chromatin domains [33]. These 53BP1 NBs are thought to promote genome stability by sequestering unrepaired DNA lesions away from nucleases in G1/early S phase until appropriate repair events and subsequent 53BP1 NB dissolution in late S phase [34, 35]. Notably, 53BP1 NBs typically divide symmetrically between sister-daughter cells, suggesting equal segregation of mitotic DNA lesions to daughter cells [33, 41].

2.1. Origin and composition of 53BP1 NBs

Unlike most NBs, which include transcriptional machinery components [38], 53BP1 NBs are transcriptionally silent and lack the initiating and elongating forms of RNA Polymerase II (Pol II) [34, 43]. Instead, they contain factors involved in DSB repair signaling (e.g. ATM, γH2AX, NBS1, MDC1, and the ubiquitin ligases RNF8 and RNF168) (Fig. 1). This composition suggests that 53BP1 NBs accumulate at DNA damage sites. RS factors (e.g. FANCD2, RPA, ATR, and ATRIP) are largely absent from 53BP1 NBs [33], although loss of either homologous recombination (HR) repair (e.g. BRCA2, PALB2, RAD51, FANCD2, and BLM) [33, 44] or replication stress factors (MCM10, TopBP1, Topoisomerase 2A, ATR, and ATRIP) promote the formation of 53BP1 NBs, highlighting their dynamic nature in response to various DNA lesions.

Fig 1: Cell cycle-dependent formation and composition of 53BP1 NBs.

Under-replicated DNA (UR-DNA) from the previous S phase (mother cell) induces the formation of 53BP1 NBs in G1 daughter cells. 53BP1 NBs are transcriptionally silent but consist of various DNA damage signaling factors (e.g. MDC1, MRN, ATM, BRCA1, RNF8, and RNF168, as shown in the figure), and chromatin compaction factors (e.g. HP1, CTCF, and SPOC1, as shown in the figure). These factors are thought to function to sequester unrepaired DNA lesions away from various nucleases until appropriate repair of these lesions, followed by dissolution of 53BP1 NBs in the late S phase. Abbreviations: Ub, Ubiquitin; MRN, MRE11-RAD50-NBS1; CTCF, CCCTC-binding factor; HP1, Heterochromatin protein 1. Created with Biorender.com.

Exogenous RS generates distinct patterns of DNA alterations [45], and 53BP1 NBs respond selectively to these stresses. For example, low dose aphidicolin (APH) treatment causes replication stress at common fragile sites (CFS), which promotes 53BP1 NBs formation [33, 34]. In contrast, hydroxyurea (HU)-induced replication stress does not cause CFS instability and consequently does not induce 53BP1 NBs [34, 46]. This selectivity stems from the propensity of CFSs for double fork stalling (DFS) events, which are the primary source of UR-DNA. 53BP1 NBs localize most highly to DNA lesions arising from UR-DNA generated in a previous S phase [47, 48]. In conclusion, 53BP1 NBs exhibit dynamic responses to specific DNA lesions arising from replication stress, adapting their formation and composition to maintain genomic stability.

2.2. Replication stress and 53BP1 NBs formation

Alterations in the number of active replication origins affects 53BP1 NB formation [41, 49]. For example, depletion of the replicative helicase components MCM4/5 or the replication licensing factor CDT1 leads to UR-DNA, thereby promoting 53BP1 NBs accumulation. Conversely, overexpressing the pre-replicative complex component CDC6 decreases the number of 53BP1 NBs [41], suggesting a specific 53BP1-dependent sensing mechanism of under-replicated DNA regions between stalled forks. Although RS leads to the formation of ssDNA regions, 53BP1 NBs rarely localize with the ssDNA-binding protein RPA, with less than 1% of 53BP1 NBs containing RPA in response to low dose APH treatment [33, 34]. However, this dynamic is shifted upon reduction of active replication origins through partial depletion of MCM2–7, increasing the percentage of RPA co-localization with 53BP1 NBs to 30% in G1 phase of the cell cycle [41]. This is thought to occur due to UR-DNA regions that are unwound during mitosis, exposing ssDNA regions which are subsequently partially coated by RPA and transmitted to daughter cells. For example, depletion of MCM5 leads to a significant increase in early-mitotic EdU incorporation that co-localizes with 53BP1 and RPA without an increase in γH2AX foci, suggesting an active DNA synthesis in UR-DNA regions during early mitosis [41].

2.3. Common Fragile Sites (CFSs) and 53BP1 NBs

53BP1 NBs arise in response to specific types of replication stress and most commonly form at CFSs induced by low dose APH [33, 34, 45]. CFSs are extensive chromosomal regions that frequently develop secondary AT-rich structures, impeding replication fork progression. This can lead to fork stalling and eventual DNA breakage, observable as gaps or breaks on metaphase chromosomes [50]. Many chromosomal rearrangements present in human cancers originate from CFSs [51, 52]. The DNA structure-specific nuclease MUS81-EME1 has been shown to maintain genome integrity at CFSs during replication stress by cleaving unreplicated CFSs, facilitating sister chromatid disjunction during mitosis [48]. In the absence of MUS81-EME1, the CFSs remain unbroken during mitosis, resulting in intertwined regions between unreplicated parental DNA strands that hinder proper sister chromatid disjunction. This leads to the formation of ultrafine bridges (UFBs) or anaphase bridges, which, if unresolved, trigger chromosome mis-segregation events and distribute DNA unevenly between daughter cells [48]. As a result, 53BP1 NBs form around the UR-DNA derived from these CFS loci.

Polymerases associated with the maintenance of CFS stability have also been shown to influence 53BP1 NB dynamics. Depletion of Rev3L, the catalytic subunit of polymerase zeta (Pol ζ), causes CFS instability [53]. Similarly, the absence of polymerase eta (Pol η) during replication stress leads to UR-DNA and subsequent 53BP1 NB formation [54]. Both hyper-recruitment of polymerase kappa (Pol κ) to replication forks or its loss have both been shown to induce 53BP1 NBs [55, 56], indicating that precise regulation of specific polymerases is necessary for preventing 53BP1 NB formation. Additionally, factors that regulate CFS-associated polymerases have also been shown to affect 53BP1 NB dynamics: the ubiquitin and SUMO ligases RAD18 and PIAS1, respectively, prevent chromosomal aberrations in mitosis by promoting polymerase eta recruitment to CFSs, thereby reducing 53BP1 NBs [57]. Overall, these studies illustrate that the regulation of 53BP1 NBs at CFSs is tightly controlled by several specialized polymerases and their associated regulators.

2.4. Cell cycle-dependent repair of UR-DNA by 53BP1 NBs

53BP1 nuclear bodies (NBs) exhibit a unique characteristic among NBs: their confinement to the G1 phase of the cell cycle, where they coordinate DNA repair spatially and temporally [33–35, 58]. As cells transition into S phase, 53BP1 NB levels gradually decline, with most resolving by late S phase. This pattern suggests the involvement of cell cycle-specific factors in regulating 53BP1 NB function [35]. Prior to dissolution, 53BP1 NBs co-localize with active replication sites (EdU- and PCNA-positive regions), indicating that active replication is essential for their repair and dissolution. This is corroborated by observations that inhibiting new origin firing impairs 53BP1 NB repair and dissolution [35].

Furthermore, S phase 53BP1 NBs contain Rap-1-interacting factor 1 (RIF1), an evolutionarily conserved genome maintenance protein that suppresses premature firing of late origins [59–62]. In concert with RIF1 and the Shieldin complex, 53BP1 channels UR-DNA from the previous S phase into replication-coupled RAD52-dependent repair (BIR-like mechanism), while restricting RAD51-dependent recombination [35, 63]. While RAD51-dependent recombination occurs behind the replication fork when a sister chromatid is available, UR-DNA-derived lesions are located ahead of the incoming replication fork, necessitating RAD52-dependent BIR-like repair [35] (Fig. 2). RAD52 loss leads to defective dissolution of 53BP1 NBs, while RIF1 or Shieldin loss increases genotoxic RAD51-mediated recombination of UR-DNA lesions, leading to chromosomal aberrations in subsequent mitosis [35]. Thus, by regulating replication timing and repair pathway choice at UR-DNA loci, 53BP1 NBs ensure accurate UR-DNA genome duplication and prevent genomic instability from genotoxic repair events.

Fig 2: Repair outcomes for UR-DNA lesions and formation of 53BP1 NBs.

UR-DNA regions in S phase arise from unresolved replication intermediates or incomplete replication caused by both endogenous stresses (e.g. CFS, large replicons, changes in the number of replication origins) and exogenous stresses (e.g. APH-induced replication stress). Mitotic entry with UR-DNA leads to DNA lesions, initiating mitotic DNA synthesis (MiDAS), where the MUS81-EME1 structure-specific endonuclease together with its scaffold protein SLX4 cleave under-replicated DNA loci as part of a RAD52-dependent repair mechanism. Successful channeling of mitotic lesions into RAD52-dependent BIR-like repair leads to 53BP1 NB dissolution. Additionally, the BLM-Topo III-RMI1-RMI2 (BTR)-TOP2 complex in MiDAS prevents the formation of bulky anaphase bridges that would otherwise lead to inefficient repair of mitotic lesions and subsequent genome instability. The direct link between bulky anaphase bridges and 53BP1 NB formation is unclear and warrants further investigation. Mitotic DNA lesions can also result in ultra-fine bridges (UFBs) that are prevented by the BTR-PICH DNA helicase complex. Aberrant dissolution of UFBs induces the formation of 53BP1 NBs that are either dissolved in the next S phase, or lead to cell quiescence if unresolved. Abbreviations: UR-DNA, under-replicated DNA; CFS, common fragile sites; APH, aphidicolin. Created with Biorender.com.

Various DDR kinases and phosphatases influence 53BP1 NB dynamics throughout the cell cycle [64–68]. The mitotic kinases polo-like kinase 1 (PLK1) and cyclin-dependent kinase 1 (CDK1) phosphorylate 53BP1, preventing its chromatin recruitment during mitosis [65, 67]. Conversely, the PP4-phosphatase complex dephosphorylates 53BP1 to permit 53BP1 NB formation at the onset of G1 [66, 68]. Additionally, the replication initiation factor TopBP1 forms distinct foci after mitotic entry in response to replication stress that disappear during mitosis and gradually transition into G1 53BP1 NBs [69]. These studies highlight the complex, dynamic cell cycle-dependent regulation of 53BP1 NB formation. Beyond cell cycle-dependent factors, 53BP1 NB dissolution can also occur due to replication-coupled dilution of the H4K20me2 histone mark in S phase [70]. However, the mechanistic underpinnings of 53BP1 NB dissolution in different contexts warrants further investigation.

2.5. Mitotic DNA Synthesis (MiDAS) and 53BP1 NBs

Mitotic DNA synthesis (MiDAS) has been characterized as a mechanism that resolves replication failures that occur at intact replication forks that fail to converge before the onset of mitosis. MiDAS most commonly occurs at difficult to replicate DNA regions, such as CFSs – sites where 53BP1 NBs tend to accumulate. Upon RS, sister chromatids from fragile sites are interlinked by replication intermediates and bound by the Fanconi anemia (FA) protein FANCD2 [71, 72]. The MUS81-EME1 structure-specific endonuclease and its scaffold protein SLX4 then promote POLD3-dependent DNA synthesis at CFSs, thereby decreasing chromosome non-disjunction and mis-segregation [73]. Crucially, MiDAS and 53BP1 NB formation are the two main post-replicative processes that control the consequences of UR-DNA carried over from a previous S phase. Furthermore, 53BP1 NB formation serves as a readout to identify factors necessary in MiDAS; failure to undergo MiDAS in response to RS channels UR-DNA into 53BP1 NBs (Fig. 2) [74]. For example, loss of SLX4 or failure to recruit MUS81 to chromatin have both been shown to induce of 53BP1 NB formation [71]. However, the precise mechanistic interplay between MiDAS and 53BP1 NBs is still not fully understood. The absence of specific MiDAS proteins increases the formation of 53BP1 NBs, suggesting that these NBs act as a backup mechanism when MiDAS fails. Alternatively, it is possible that UR-DNA is initially counteracted by activating dormant origins coupled with cell cycle delays to facilitate replication completion within a given S phase, a process mediated by 53BP1 NBs. When this approach fails, cells then resort to MiDAS. From a genome stability perspective, this could be more advantageous as a considerable fraction of UR-DNA escapes MiDAS and leads to the formation of UFBs, chromosomal instability, and genomic rearrangements [35, 71, 72, 75, 76]. Further studies are needed to determine in which cellular contexts cells prefer 53BP1 NB-mediated resolution of UR-DNA over MiDAS, and to elucidate the molecular dynamics between 53BP1 NBs and MiDAS at specific genomic loci.

3. Regulation of 53BP1 in S/G2 phase

53BP1 is an oligomeric chromatin reader that binds a combination of methylated and ubiquitinated histones (H4K20me2 and H2AK15Ub) within DSB-associated chromatin [6, 64, 77]. Although the S/G2 phase is a favorable environment for BRCA1 and HR, 53BP1 can still be recruited to breaks in S/G2 cells, albeit with reduced efficiency due to the dilution of H4K20me2 on chromatin by replication [70, 78]. As in G1, RIF1 is recruited to DSBs in S/G2 via ATM-dependent phosphorylation of 53BP1. Once recruited, 53BP1 and RIF1 form a barrier against end resection by MRN and CtIP, thus preventing resection. In G1 or early G2, 53BP1 localizes to breaks and forms ionizing radiation-induced foci (IRIF). However, in late G2, these IRIF enlarge, with 53BP1 displaced from the periphery while RPA occupies the core [79]. The removal of 53BP1 in S/G2 relies on BRCA1-dependent recruitment of the PP4C phosphatase, which dephosphorylates 53BP1 and releases RIF1 from chromatin. [80, 81]. Additionally, the E3 ubiquitin ligase SPOP has been shown to exclude 53BP1 from chromatin. In response to DNA damage, ATM kinase–catalyzed phosphorylation of SPOP causes a conformational change in SPOP that stabilizes its interaction with 53BP1. This interaction induces polyubiquitination of 53BP1 in S phase, eliciting 53BP1 extraction from chromatin by a valosin-containing protein/p97 segregase complex [82]. However, in response to RS, 53BP1 has been shown to be recruited to breaks via ATMIN dependent ATM-mediated signaling [33, 34, 83, 84]. This recruitment is independent of NBS, as reduction of NBS results in increased 53BP1 foci, both in basal conditions and after aphidicolin treatment. This competition model of ATMIN and NBS for ATM binding shows the specificity of ATMIN for ATM activation following replicative stress and for NBS following the induction of DNA double-strand breaks [83, 85].

4. Replication Fork Dynamics

DNA replication is initiated at replication origins, and involves the replisome—a complex of DNA helicases and polymerases—unwinding template DNA and synthesizing new strands [86–90]. Despite its accuracy, DNA replication faces challenges from various factors, leading to replication stress (RS) [91, 92]. RS can slow fork progression, decrease fidelity, and cause DNA breaks, threatening genome stability [93–95]. Common causes of RS include oncogene activation, nucleotide depletion, transcription-replication conflicts, and difficult-to-replicate regions [92, 96]. In response, cells activate multiple pathways to stabilize, repair, and restart replication forks, ensuring accurate genome duplication and maintaining stability.

4.1. Fork Stalling and Restart

53BP1 also prevents nascent strand degradation by MRE11 [9, 10] (Fig. 3b) and competes with BRCA1 for stalled fork restart. 53BP1 and RIF1 promote a fast, fork-cleavage-independent restart pathway, while BRCA1 facilitates a slower, break-induced replication (BIR) pathway that involves fork cleavage by SLX4-MUS81 [8].

Fig 3: Multifunctional role of 53BP1 in replication fork stability and protection.

(a) 53BP1 recruitment at stalled replication forks enhances the ATR/CHK1 signaling pathway in response to replicative stress. (b) 53BP1 protects forks from nascent strand degradation by MRE11. (c) TPX2 forms a complex with Aurora A to inhibit 53BP1 chromatin accumulation, which protects newly replicated DNA from MRE11 nuclease degradation. (d) 53BP1 is an RNA-binding protein that directly interacts with RNA-DNA chimeric structures. Depletion of 53BP1 perturbs the maturation of Okazaki fragments. Figure adapted from [29], [31], and [113]. Created with Biorender.com.

Given 53BP1’s critical role in controlling resection at DSBs, its function at stalled replication forks has been explored extensively. Proteomic datasets reveal 53BP1 presence at replication forks, with colocalization observed in cells experiencing replication stress [25, 26]. RNF168, a DDR factor which is necessary to modify the chromatin landscape for 53BP1 localization during DSB repair, is also necessary for 53BP1 recruitment at sites of unperturbed DNA replication in S phase [28]. 53BP1-deficient cells are hypersensitive to replication stress, exhibit defective fork restart after an acute replication challenge, have decreased replication rates, and increased fork reversal in the absence of added replication stress [27–29, 97]. Lo et al. showed that while 53BP1 colocalizes with EdU after HU treatment, SMARCAD1 prevents its toxic accumulation at active or restarted forks. Without SMARCAD1, 53BP1 mediates untimely PCNA dissociation via the PCNA-unloader ATAD5, leading to frequent fork stalling, inefficient restart, and the buildup of single-stranded DNA [98].

Besides its role in p53 signaling and activation of the G1/S checkpoint [24], 53BP1 recruitment at stalled replication forks enhances the ATR/CHK1 signaling pathway in response to replicative stress [29] (Fig. 3a). Yoo et al. observed that 53BP1 forms a complex with RPA1 and RPA2, two components of the replication protein A (RPA) complex, which was disrupted upon camptothecin treatment. RPA2 hyperphosphorylation after camptothecin treatment was inhibited in cells expressing dominant-negative fragments of 53BP1 (either the amino- or carboxy-terminal of 53BP1 which exerts dominant-negative effects on 53BP1 function), implying that 53BP1 is required for RPA2 phosphorylation by acting as a mediator for recruiting upstream kinases such as ATM or DNA-PK to phosphorylate RPA2 [99]. 53BP1 protects forks from MRE11-mediated nascent strand degradation [28, 29] (Fig. 3b). It competes with BRCA1 for fork restart, promoting a fast, cleavage-independent pathway with RIF1, while BRCA1 facilitates a slower break-induced replication (BIR) pathway involving SLX4-MUS81 [27].

DNA end-resection is an early key step in HR that is controlled by BRCA1 and 53BP1 proteins. While 53BP1 restricts the formation of recombinogenic single-stranded DNA (ssDNA) at canonical DSBs, the loss of 53BP1 can restore end resection and HR in BRCA1-deficient cells [100–102]. Importantly, beyond faciltitating DNA end resection BRCA1 also promotes HR by loading RAD51 onto ssDNA to initiate strand invasion. However, in the context of collapsed replication forks, a study by Pavani et al. found that BRCA1 deficiency did not alter DNA end resection, although RAD51 loading was diminished; notably, the absence of 53BP1 restored RAD51 loading in this scenario [103]. Thus, unlike at canonical DSBs, where 53BP1 impacts end resection, at collapsed forks it primarily limits RAD51 filament formation. These results highlight a distinct regulatory role for these proteins depending on the type and the context of the DNA lesion.

4.2. Fork Protection

Despite many studies concluding that loss of 53BP1 and associated NHEJ factors restores HR in BRCA1-deficient cells, 53BP1 loss is not believed to function in replication fork protection [101, 104]. However, TPX2, a 53BP1 interacting protein was recently shown to function in protecting stalled replication forks [31, 105]. TPX2 forms a complex with Aurora A to inhibit 53BP1 chromatin accumulation, thereby preventing MRE11-mediated degradation of newly replicated DNA (Fig. 3c). This fork protection is likely due to the presence of TPX2 and Aurora A in replisomes. They further show that TPX2 and BRCA1 exist in separate fork protection pathways, as 53BP1 depletion rescued fork degradation due to TPX2 loss, but did not rescue in BRCA1 deficient cells [31, 104]. These results reveal an unappreciated role for 53BP1 in fork protection.

In addition, 53BP1 has been shown to suppress DNA2-mediated nascent strand degradation in BRCA1 proficient cells [32, 106]. However, this role for 53BP1 in fork protection was found to not be universally conserved, but dependent on cellular context and the nature of 53BP1 inactivation. Liu et al. found that elevated γH2AX and replication stress sensitivity of 53BP1 deficient cells is dependent on FBH1, a helicase that has been found to catalyze replication fork reversal [107], suggesting that protection of nascent DNA from nucleases after FBH1-dependent fork remodeling is a key function of 53BP1 at stalled forks. These findings highlight 53BP1’s complex role in maintaining replication fork stability, reconciling seemingly contradictory observations across different studies [28, 29, 31, 108].

5. Okazaki Fragments

Recent observations highlight the importance of RNA-binding proteins (RBPs) in genome maintenance. The accumulation of 53BP1 at DSBs is RNA dependent [109–111], and RNA immunoprecipitation (RIP) assays have shown that 53BP1 associates with RNAs [110, 111]. A recent proteomic analysis has identified 53BP1 as a candidate protein interacting with RNA [112]. Additionally, Leriche et al. utilized UV-C-induced crosslinking IP (CLIP) and complex capture (2C) to provide evidence that 53BP1 directly interacts with RNA [113]. They further show that 53BP1 directly interacts with Okazaki fragments in the absence of external stress (Fig. 3d). The recruitment of 53BP1 to nascent DNA shows susceptibility to in situ ribonuclease A treatment and is dependent on PRIM1, which synthesizes the RNA primer of Okazaki fragments. Conversely, depletion of FEN1, resulting in the accumulation of uncleaved RNA primers, increases 53BP1 levels at replication forks, suggesting that RNA primers contribute to the recruitment of 53BP1 at the lagging DNA strand. 53BP1 depletion induces an accumulation of S-phase poly (ADP-ribose), which constitutes a sensor of unligated Okazaki fragments [113]. These data indicate that 53BP1 is anchored at nascent DNA through its RNA-binding activity, highlighting the role of an RNA-protein interaction at replication forks.

6. Conclusions

53BP1 plays important functions at the replication fork under both stressed and normal conditions. During replication stress, 53BP1 contributes to fork protection by regulating fork reversal and restart. It helps maintain replication fork stability and speed, with 53BP1 loss leading to defective fork progression and increased sensitivity to replication stress agents. However, 53BP1’s precise role in fork protection remains debated, as some studies have not observed protective effects. Under non-stressed conditions, 53BP1 interacts with Okazaki fragment RNA primers during DNA replication. Additionally, 53BP1 NBs play a critical role in shielding under-replicated DNA in G1 phase to prevent genomic instability. The formation of these NBs allows 53BP1 to regulate replication timing of difficult-to-replicate genomic regions in the subsequent S phase. While 53BP1 loss can restore HR in BRCA1-deficient cells, its replication fork protection functions appear context-dependent. Further research is needed to fully elucidate the mechanisms and conditions under which 53BP1 acts to maintain genome stability during DNA replication.

Highlights.

• Formation of 53BP1 nuclear bodies allows 53BP1 to regulate replication timing of difficult-to-replicate genomic regions in the subsequent S phase.

• 53BP1 regulates fork reversal and restart, and maintains replication fork stability and speed.

• Unlike at canonical DSBs where 53BP1 impacts end resection, 53BP1 primarily limits Rad51 filament formation at collapsed forks.

• Under normal conditions, 53BP1 is anchored on lagging strand replication forks through its interaction with Okazaki fragment RNA primers.

Abbreviations

2C

complex capture

APH

aphidicolin

BIR

break-induced replication

BRCT

BRCA-carboxyterminal

CTCF

CCCTC-binding factor

CFS

common fragile site

CLIP

UV-C-induced crosslinking immunoprecipitation

CSR

class-switch recombination

DFS

double fork stalling

DSBs

double-strand breaks

FA

Fanconi anemia

FFR

focus forming region

GAR

glycine-arginine rich

HP1

Heterochromatin protein 1

HR

homologous recombination

HU

hydroxyurea

IRIF

ionizing radiation-induced foci

MiDAS

mitotic DNA synthesis

MOB

mobility domain

NBs

nuclear bodies

NHEJ

non-homologous end joining

NLS

nuclear localization signal

OD

oligomerization domain

OPT

Oct1/PTF/transcription

RBP

RNA-binding protein

RIP

RNA immunoprecipitation

RS

replication stress

ssDNA

single-stranded DNA

Ub

ubiquitin

UDR

ubiquitin-dependent recruitment motif

UFBs

ultrafine bridges

UR-DNA

under-replicated DNA

Data Availability

No data was used for the research described in the article.