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. Author manuscript; available in PMC: 2007 Dec 29.
Published in final edited form as: Mol Cell. 2007 Nov 9;28(3):482–490. doi: 10.1016/j.molcel.2007.08.027

Promotion of Homologous Recombination and Genomic Stability by RAD51AP1 via RAD51 Recombinase Enhancement

Claudia Wiese 1,*, Eloïse Dray 2,*, Torsten Groesser 1, Joseph San Filippo 2, Idina Shi 2, David W Collins 1,4, Miaw-Sheue Tsai 1, Gareth Williams 1, Bjorn Rydberg 1, Patrick Sung 2,3, David Schild 1,3
PMCID: PMC2169287  NIHMSID: NIHMS34042  PMID: 17996711

Summary

Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand pairing step in HR. RAD51AP1 (RAD51 Associated Protein 1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress, and RAD51AP1 is epistatic to the HR protein XRCC3. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA damaging treatment. Purified RAD51AP1 binds both dsDNA and a D-loop structure, and, only when able to interact with RAD51, greatly stimulates the RAD51-mediated D-loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.

Introduction

Homologous recombination (HR) represents a major mechanism for the elimination of DNA double-strand breaks (DSB)s, inter-strand DNA crosslinks, and other types of deleterious chromosome lesions. HR that occurs early in meiosis is essential for tying homologous chromosomes together until time for their segregation in the first meiotic division (Symington, 2002; Sung and Klein, 2006). Interestingly, HR also plays a role in telomere maintenance (McEachern and Haber, 2006). By virtue of its involvement in the repair of DNA damage and in other chromosome transactions, HR is indispensable for genomic integrity. Accordingly, dysfunction in HR leads to chromosome fragility and, in humans, the cancer phenotype (Jasin, 2002; Surralles et al, 2004; Sung and Klein, 2006).

HR in mitotic cells is catalyzed by RAD51. Like its prokaryotic ortholog RecA, RAD51 polymerizes onto ssDNA to form a helical filament, called the presynaptic filament, and it catalyzes D-loop formation within the context of this filament. The recombinase activity of RAD51 is regulated by various protein-protein interactions (Symington, 2002; Sung and Klein, 2006).

RAD51AP1 (HUGO designation for PIR51) is a vertebrate-specific RAD51-interacting protein (Kovalenko et al., 1997; Mizuta et al., 1997). Here, we show an involvement of RAD51AP1 in HR and the homology-directed repair of chromosome damage. Importantly, RAD51AP1 is found to interact with and greatly enhance the recombinase activity of RAD51. Together, the genetic, cytological, and biochemical data presented herein establish RAD51AP1 as an HR factor that exerts its biological function through physical and functional interactions with RAD51.

Results

RAD51AP1 Knockdown Causes DNA Damage Sensitivity and Chromatid Breaks

We used three different shRNAs to deplete RAD51AP1 (Fig. 1 and Fig. S1A-C). HeLa cells treated with these RAD51AP1-targeting shRNAs were more sensitive (∼2.3-fold based on D10 values) to mitomycin C (MMC) than cells not treated with any shRNA or with GFP-targeting shRNA or mutated shRNA #2 (Fig. 1A). The MMC-sensitivity of RAD51AP1-depleted HeLa cells is complemented by introducing the EGFP-RAD51AP1res that is resistant to shRNA #2 (Fig. 1B and Fig. S1D). Because of the transient expression/stability of the EGFP-RAD51AP1 (here: EGFP-RAD51AP1res) fusion protein, it was necessary to perform the complementation in a two-step process (see the Supplemental Data). The apparent lack of complete complementation likely is due to cells transfected with the shRNA #2 plasmid that were not subsequently transfected by EGFP-RAD51AP1res. Simultaneous depletion of both RAD51AP1 and the RAD51 paralog XRCC3 results in the same cellular sensitivity to MMC as depletion of RAD51AP1 alone, suggesting that RAD51AP1 and XRCC3 work in the same biological pathway (Fig. 1C). Although defects in HR frequently result in mild growth retardation (Hinz et al., 2006), shRNA-targeted depletion of RAD51AP1, XRCC3 or both did not result in a significant reduction in the plating efficiency of HeLa cells (Fig. S2B).

Figure 1. Induction of MMC, CPT and X-ray Sensitivity and Chromatid Breaks by RAD51AP1 Knockdown.

Figure 1

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(A) Survival curves of RAD51AP1-depleted HeLa cells treated with MMC. Non-depleting negative controls: pRNAi, GFP shRNA and mutated shRNA #2. All points with error bars in panels A-E represent the average of at least three different experiments ± 1 SD. * Note: shRNA #1 was tested in triplicate but with 2 µM MMC only, and resulted in the same sensitization as shRNA #2 and #3.

(B) Complementation of RAD51AP1-depletion in HeLa cells. Cells expressing both shRNA #2 and EGFP-RAD51AP1res are more resistant to MMC than cells expressing shRNA #2 only.

(C) Epistasis between Rad51AP1 and XRCC3. HeLa cells depleted for both XRCC3 and RAD51AP1 show the same sensitivity to MMC as cells depleted for RAD51AP1 only.

(D and E) Survival curves of RAD51AP1-depleted asynchronous HeLa cells treated with camptothecin or X-rays. Non-depleting negative controls are GFP shRNA and mutated shRNA #2 for D, or just mutated shRNA #2 for E. Note: shRNAs #1 and #3, which do not sensitize asynchronous HeLa cells to X-rays, do sensitize cells synchronized in S-phase (Fig. S2A).

(F) Representative western blot analysis to show the extent of RAD51AP1-depletion observed in HeLa cells with three different shRNAs; probed with anti-GFP antibody. The EGFP-RAD51AP1 fusion protein is a surrogate marker for RAD51AP1 expression. pRNAi is a negative control and QM (a transcription factor) is a loading control. Additional western blots, also demonstrating depletion of endogenous RAD51AP1, are presented in Figs. 2, S1 and S2.

(G and H) MMC (50nM)-induced and spontaneous chromatid breaks in RAD51AP1-depleted cells. Mutated shRNA #2 is a non-depleting negative control. In panel H, spontaneous ctbs were scored 72 h and 96 h after RAD51AP1-depletion. Error bars: ± 1 SE for two (G) or three (H) independent experiments.

(I) Giemsa-stained metaphase spread of MMC-treated RAD51AP1-depleted HeLa cells. Black arrows: chromatid breaks; red arrow: chromatid-type exchange aberration.

Since RAD51AP1-depleted cells exhibit sensitivity to MMC, we tested whether RAD51AP1 is involved in the Fanconi anemia (FA) pathway. Most of the FA proteins are required for the mono-ubiquitination of FANCD2 after DNA damage. Depletion of RAD51AP1 has no effect on FANCD2 mono-ubiquitination in HeLa cells (Fig. S1E), although a role of RAD51AP1 in the FA pathway cannot be ruled out at present.

RAD51AP1 knockdown with either shRNA #1, #2 or #3 sensitizes cells to the topoisomerase I poison camptothecin (CPT) (∼1.6-fold; Fig. 1D), a compound that leads to double-strand breaks at the replication fork. Only one of these three shRNAs (shRNA #2) resulted in increased sensitivity of asynchronous HeLa cells to X-rays (Fig. 1E). Because no significant difference in the extent of RAD51AP1-depletion was observed treating with shRNA#2 or shRNA#3 (Fig. 1F and Fig. S1B), an off-targeted effect for shRNA#2 on X-ray sensitivity remains a possibility. Because shRNA #1 or shRNA #3 did not result in detectable X-ray sensitization (Fig. 1E), their effects on X-irradiated and synchronized HeLa cells were assessed. Indeed, mild sensitivity to X-rays was seen upon treatment of HeLa cells with shRNA #1 or shRNA #3 in S-phase (Fig. S2A). This result is consistent with our previous observation for RAD51C-depleted HeLa cells in S-phase (Lio et al., 2004).

RAD51AP1-depleted cells were tested for their levels of chromatid breaks (ctbs), both spontaneously and after DNA damage. Following one acute 1 h exposure to 50 nM MMC (Fig. 1G gray bars), a 4- to 6-fold increase in ctbs was observed for RAD51AP1-depleted cells. In addition, significantly more spontaneous ctbs were present in the RAD51AP1-depleted cells (Fig. 1G white bars, and 1H). Following MMC treatment, we observed a 4- to 5-fold increase in chromatid-type exchange aberrations involving two or more chromosomes in cells upon RAD51AP1 knockdown (∼0.5% in control cells, and ∼2.5% and ∼2.3% in cells depleted for RAD51AP1 by shRNA #2 and #3, respectively (Fig. 1I)). These chromatid aberrations likely arise from non-homologous end-joining events involving breaks on different chromosomes. Taken together, our results show that RAD51AP1 is epistatic to XRCC3 and necessary for DNA damage repair and/or response in S-phase cells.

Involvement of RAD51AP1 in HR

We used a cell line harboring the DR-GFP recombinational reporter (Wiese et al., 2002b) to query whether RAD51AP1 has a role in HR. The reporter harbors a direct repeat of the mutant green fluorescent protein (GFP) gene. A site-specific DSB, generated in the upstream GFP copy by the I-SceI endonuclease, triggers gene conversion with the downstream GFP copy to yield wild type GFP; flow cytometry was used to analyze the recombinant green fluorescent cells. TK6-neo-DRGFP cells were co-transfected with the I-SceI expression plasmid and plasmids encoding shRNAs targeting either RAD51AP1 or XRCC3. As negative controls, we used a plasmid that codes for mutated shRNA #2 (which, as shown in Fig. S1C, has no effect on RAD51AP1 expression) and another plasmid that does not code for any shRNA. Reproducibly, knockdown of RAD51AP1 by either of two different shRNAs results in an ∼2- to 2.5-fold decrease in HR (Fig. 2A and Fig. S3C), similar to what we observed for the knockdown of XRCC3 (Fig. 2A), which is known to be involved in HR (Pierce et al., 1999). The 2- to 2.5-fold decrease in HR that we observe is comparable to that reported for siRNA-depletion of RAD51C (another RAD51 paralog) (Lio et al., 2004).

Figure 2. RAD51AP1-depletion Impairs Homologous Recombination but not RAD51 DNA Repair Foci.

Figure 2

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(A) TK6-DRGFP cells depleted for RAD51AP1 (shRNA #2 or shRNA #3) or XRCC3 show ∼2-to 2.5-fold lower levels of GFP+ cells (i.e. homologous recombinants) than control cells transfected with either pRNAi or mutated shRNA #2. In four experiments, both negative controls were used and both gave very similar numbers (for example, see Fig. S3C). Therefore, their values were averaged and this average was set to 1.0. In the remaining experiments only mut. shRNA was used as a negative control and this number was set at 1.0. Data for the depleted cells are the mean of the relative fraction of GFP+ cells from 5-7 independent experiments ± 1 SE (see also Fig. S3C).

(B and C) Western blot analyses to show the extent of EGFP-RAD51AP1 (here: AP1)-depletion (B) or XRCC3 (here: X3)-depletion (C) in TK6-DRGFP cells. Two hairpins were tested for each gene (lanes 2 and 3 corresponding to shRNA #2 and shRNA #3, respectively for RAD51AP1) and compared to control cells (lanes 1: transfected with mutated shRNA #2). QM: loading control.

(D) RAD51 foci form normally in RAD51AP1-depleted HeLa cells after 8 Gy X-rays. Control transfected HeLa cells (mutated shRNA #2; panels A and B) and RAD51AP1-depleted cells (panels C-F) display bright RAD51 foci in ∼ 80 % of the cells analyzed. In contrast, HeLa cells depleted for either RAD51C (panels G and H) or XRCC3 (panels I and J) show impaired RAD51 foci formation (i.e. only some cells form overall smaller and less intense RAD51 foci). The single cell with large foci in panel J may not have been transfected with the XRCC3 shRNA. Cells were fixed and stained at 8 h after 8 Gy X-rays. Two panels for each sample from different areas of the chamber slide are shown.

(E) Western blot analysis demonstrating the depletion of RAD51AP1, XRCC3 or RAD51C for the experiment shown in panel D. RAD51 acts as both a non-depleted negative control and as a loading standard. XRCC3 and Rad51C exist as a complex (Wiese et al., 2002a), and depletion of XRCC3 reduces the level of RAD51C and vice versa, as has previously been reported (Lio et al., 2004).

RAD51AP1 Interacts with RAD51 In Vivo

We found that upon X-ray treatment, ectopically expressed EGFP-RAD51AP1 co-localizes with RAD51 nuclear foci in U2OS cells, further supporting an involvement of RAD51AP1 in HR (Fig. S3A). Using EGFP-RAD51AP1 and HA-RAD51, and nuclear cell extracts from transfected HeLa cells, we found a co-immunoprecipitable complex of RAD51AP1 and RAD51 (Fig. S3B).

RAD51AP1 Depletion Has No Effect on RAD51 Focus Formation

We asked whether X-ray-induced RAD51 focus formation is attenuated in cells with reduced levels of RAD51AP1, as some HR mutants show an impairment in this regard (Yuan et al., 1999; Bishop et al., 1998; van Veelen et al., 2005). However, whereas X-ray-induced RAD51 focus formation is reduced in HeLa cells depleted for either RAD51C or XRCC3, X-ray- and MMC-induced RAD51 foci assemble normally in HeLa cells depleted for RAD51AP1 (Fig. 2D and Fig. S3D, and data not shown).

RAD51AP1 Purification

We added a cleavable GST-tag and a (His)6-tag to the amino and carboxyl termini of RAD51AP1, respectively, and expressed the tagged protein in E. coli. The tagged RAD51AP1 is soluble, and a procedure was devised for its purification to near homogeneity (Fig. 3A). The GST portion of the purified protein could be removed using the PreScission protease, and the cleaved protein was purified and used in the DNA binding studies and initial experiments that explored functional interactions with RAD51. However, we have found that the GST tag does not affect the functional attributes of RAD51AP1 (data not shown). Likewise, the addition of an N-terminal MBP tag has no adverse effect on the functional attributes of RAD51AP1 (see later).

Figure 3. RAD51AP1 Purification and Characterization.

Figure 3

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(A) Purified GST-RAD51AP1, RAD51AP1, MBP-RAD51AP1, MBP-RAD51AP1 L319Q, MBP-RAD51AP1 H329A, and MBP-RAD51AP1 CΔ25 were analyzed by SDS-PAGE and Coomassie Blue staining.

(B) GST-tagged RAD51AP1 or GST was incubated with RAD51 or yRad51 and glutathione Sepharose beads were used to capture any protein complex that had formed. The beads were washed and treated with SDS to elute the bound proteins. The supernatant (S), wash (W) and SDS eluate (E) were analyzed by SDS-PAGE with Coomassie Blue staining.

(C) MBP-tagged wild type, L319Q, H329A, or CΔ25 RAD51AP1 protein was incubated with RAD51 and amylose agarose beads were used to capture any protein complex that had formed. The analysis was as in (B).

(D) RAD51AP1 (0.03 to 1.5 µM) was incubated with ssDNA and dsDNA (panel I) or with dsDNA and the D-loop substrate (panel III). The mobility shift of the DNA substrates was analyzed in a 10% (panel I) or 5% (panel III) polyacrylamide gel. The asterisk denotes the position of the 5′ 32P label. The results from panels I and III were plotted in panels II and IV, respectively.

Specific Interaction of RAD51AP1 with RAD51

A pulldown assay was employed to examine the ability of RAD51AP1 to interact with human RAD51. S. cerevisiae Rad51 (yRad51) and E. coli RecA were included as controls. GST-tagged RAD51AP1 was incubated with the recombinases, and glutathione Sepharose beads were used to capture any protein complex that had formed. Nearly all of the input RAD51 became associated with RAD51AP1 (Fig. 3B). The protein complex is specific, as yRad51 or RecA was not pulled down by RAD51AP1 (Fig. 3B, and Fig. S4B). The above pulldown assay was done in the presence of 100 mM KCl, and we asked whether the RAD51-RAD51AP1 complex can withstand higher salt levels. We saw little reduction in protein complex formation even at 500 mM KCl (Fig. S4A). Taken together, the results revealed a specific and stable complex of RAD51AP1 and RAD51.

RAD51-interaction defective RAD51AP1 mutants

We have used the yeast two-hybrid assay to show that the L319Q or H329A substitution in RAD51AP1 compromises the interaction with RAD51 and that deletion of the carboxyl terminal 25 amino acid residues (CΔ25) ablates complex formation with RAD51 (Kovalenko et al., 2006). To support these results, we expressed and purified to near homogeneity the wild type form, the L319Q and H329A mutant variants, and also a truncation mutant form (ΔC25) lacking the C-terminal 25 residues of RAD51AP1 that are tagged at the N-terminus with MBP and (His)6 (Fig. 3A). Using affinity pulldown on amylose agarose (to which the MBP portion of the tagged proteins binds avidly), we verified that both of the point mutants are compromised for RAD51 binding, while the ΔC25 mutant is defective in this regard (Fig. 3C).

DNA Binding by RAD51AP1

We used a DNA mobility shift assay to examine the DNA binding properties of RAD51AP1. In isolation, RAD51AP1 appeared to bind ssDNA and dsDNA equally (Kovalenko et al, 1997; data not shown). However, when RAD51AP1 was co-incubated with both DNA species, a distinct preference for dsDNA was revealed (Fig. 3D, panels I and II). We also tested the ability of RAD51AP1 to bind a D-loop substrate with a 3′ invading strand. Notably, RAD51AP1 exhibited a preference for the D-loop substrate over the dsDNA species (Fig. 3D, panels III and IV). These results are in contrast to replication protein A (RPA), which, as expected, bound ssDNA preferentially over the D-loop structure and dsDNA (Fig. S5). Thus, we have been able to verify that RAD51AP1 binds both ssDNA and dsDNA (Kovalenko et al., 1997) and have also provided evidence that it binds dsDNA preferentially and has an even higher affinity for the D-loop structure (Fig. 3D). The results support the notion that RAD51AP1 acts at a step downstream of RAD51 presynaptic filament assembly (see below).

Enhancement of RAD51 Activity by RAD51AP1

During HR, a ssDNA substrate, derived from the processing of a DSB or another DNA lesion, is utilized by the recombination machinery to invade a homologous chromosome target to yield a D-loop (Sung and Klein, 2006). We used an oligonucleotide based D-loop assay (Fig. 4A) to determine whether RAD51AP1 can enhance RAD51-mediated DNA strand pairing. Whereas only ∼2% of the input oligonucleotide was converted into D-loops by RAD51 (Fig. 4B), the addition of an amount of RAD51AP1 substoichiometric to that of RAD51 enhanced the reaction by ∼10 or more fold. RAD51AP1 alone was incapable of D-loop formation and, as expected (Sung and Klein, 2006), the reaction catalyzed by RAD51 and RAD51AP1 remains ATP-dependent (Fig. 4B). The stimulatory effect of RAD51AP1 on RAD51 is specific, as no enhancement of the D-loop reaction catalyzed by either yRad51 (Fig. S6A) or RecA (data not shown) was seen.

Figure 4. Specific Enhancement of the RAD51-mediated D-loop Reaction by RAD51AP1.

Figure 4

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(A) Schematic of the D-loop assay.

(B) D-loop reactions mediated by combinations of RAD51 (0.8 µM) and RAD51AP1 (0.05 to 1 µM). ATP was omitted from the reaction in lane 10. The results were plotted.

(C) D-loop reactions mediated by combinations of RAD51 K133R (0.8 µM) and RAD51AP1 (0.05 to 1 µM). ATP was omitted from the reaction in lane 10. The results were plotted.

(D) D-loop reactions mediated by combinations of RAD51 or RAD51 K133R (0.8 µM each) and the MBP tagged form of wild type or mutant RAD51AP1 (0.2 µM each). The results were plotted.

ATP hydrolysis by RAD51 prompts the dissociation of the presynaptic filament. Because of this, the use of a non-hydrolyzable ATP analogue (e.g. AMP-PNP) or the RAD51 K133R protein (which binds but does not hydrolyze ATP) leads to the formation of a stable presynaptic filament (Bianco et al., 1998; Bugreev and Mazin, 2004; Chi et al., 2006; Cox, 2003; Ristic et al., 2005). Importantly, a substoichiometric quantity of RAD51AP1 stimulated the D-loop forming ability of RAD51 K133R by a similar degree (Fig. 4C). When AMP-PNP was used as the nucleotide co-factor, the addition of RAD51AP1 again resulted in a marked enhancement of the D-loop reaction (Fig. S6B).

RAD51-interaction deficient RAD51AP1 mutants lack the ability to promote D-loop formation

We asked whether the RAD51AP1 mutants deficient in RAD51 binding are capable of enhancing the D-loop reaction. As expected, MBP-tagged RAD51AP1 strongly stimulated D-loop formation by either RAD51 or RAD51 K133R (Fig. 4D). Significantly, none of the RAD51AP1 mutants - L319Q, H329A, and CΔ25 - was able to enhance the reaction, regardless of whether RAD51 or RAD51 K133R was used (Fig. 4D). Because all three RAD51AP1 mutants have DNA binding properties indistinguishable from the wild type counterpart (Fig. S7), the inactivity of these proteins in the D-loop reaction very likely stems from the impairment of RAD51 interaction.

Discussion

We have shown that cells depleted for RAD51AP1 are hypersensitive to a variety of chromosomal lesions, including DNA crosslinks, topoisomerase I-DNA adducts, and X-rays. Moreover, these cells exhibit an elevated level of spontaneous and DNA damage-induced chromatid breaks. We note that a recent study also documents that RAD51AP1-depleted cells are sensitized to MMC and have elevated MMC-induced chromatid breaks (Henson et al., 2006). The pattern of DNA damage sensitivity and chromosome fragility that accompany RAD51AP1 knockdown, and the association of RAD51AP1 with RAD51, suggest a role of this protein in chromosome damage repair via HR. Cytogenetic data and a direct test for HR efficiency have provided evidence to support this premise. In addition, our results show that RAD51AP1 is epistatic with XRCC3, a RAD51 paralog known to be important for HR.

We have shown that RAD51AP1 interacts with RAD51, but, interestingly, found that it does not associate with yRad51 or E. coli RecA. Our DNA binding studies have revealed a preference of RAD51AP1 for dsDNA over ssDNA, and for D-loop over dsDNA. In an accompanying manuscript, Modesti et al (2007) also present data to show that RAD51AP1 has a high affinity for branched DNA structures. Importantly, using the D-loop assay, we show a dramatic enhancement of the RAD51 DNA strand pairing activity by RAD51AP1. Since RAD51AP1 has no effect on the recombinase activity of yRad51 and RecA, it seemed likely that its functional interaction with RAD51 was dependent on complex formation with the latter. This premise is supported by our experiments and results by Modesti et al (2007) using truncated and point mutant RAD51AP1 variants that are compromised for RAD51 binding and consequently unable to enhance D-loop formation. The biochemical results that we have presented here, in conjunction with the available genetic and cell biological data, help establish a role of RAD51AP1 in HR and homology-directed DNA repair via enhancement of the RAD51 recombinase activity.

Assembly of the RAD51 presynaptic filament is crucial to HR, as all the biochemical steps that lead to D-loop formation occur within the confines of this filament (Bianco et al., 1998; Symington, 2002; Sung et al., 2003; Sung and Klein, 2006). Cells deficient in the tumor suppressor BRCA2, any of the five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3), or SWS1 are impaired for the formation of RAD51 DNA repair foci (Symington, 2002; Martin et al., 2006). Studies involving the RAD51B-RAD51C complex, Ustilago maydis Brh2 (a BRCA2-like molecule), and a polypeptide derived from human BRCA2, have provided direct evidence that the RAD51 paralogs and BRCA2 function in the assembly of the RAD51 presynaptic filament, by aiding in the displacement of the single-strand DNA binding protein RPA from ssDNA (Sigurdsson et al., 2001; Yang et al., 2005; San Filippo et al., 2006; Sung and Klein, 2006). Our results suggest that RAD51AP1 functions at a stage in the HR reaction subsequent to the assembly of the presynaptic filament, as (1) the frequency of RAD51 DNA repair foci is not affected by RAD51AP1 knockdown, and (2) under conditions wherein the RAD51 presynaptic filament is stable, RAD51AP1 still exerts a strong stimulatory effect on the D-loop reaction. The ability of RAD51AP1 to recognize the D-loop structure may be critical for stabilizing the nascent D-loop intermediate made by RAD51.

Stimulation of the RAD51-mediated D-loop reaction has also been reported for the meiotically-expressed HOP2-MND1 heterodimer (Ploquin et al., 2007; Chi et al., 2007). The HOP2-MND1 complex enhances the ability of the RAD51 filament to capture dsDNA (Chi et al., 2007). RAD51AP1 might play a similar function during DNA repair in mitotic cells, by utilizing its dsDNA binding ability to orchestrate the capture of duplex DNA. RAD51AP1 appears to be specific to vertebrates. It is likely that vertebrates, because of their larger genome, require additional factors to ensure that HR occurs efficiently. Alternatively, non-vertebrates may possess an unidentified functional equivalent of RAD51AP1.

We note that rad54 mutants are compromised for HR and, like RAD51AP1-deficient cells, show no defect in RAD51 focus formation (van Veelen et al., 2005). The RAD54 protein possesses a robust DNA-dependent ATPase activity and it enhances the DNA strand pairing activity of RAD51 via ATP-hydrolysis driven alterations in the topology of duplex DNA (Van Komen et al., 2000; Ristic et al., 2001; Sung et al., 2003). Since RAD51AP1 does not possess any ATPase activity (data not shown), it must influence the RAD51-mediated homologous pairing reaction via a different mechanism. We do not see any evidence of RAD51AP1 and RAD54 functioning synergistically in the D-loop assay (data not shown). This lack of synergy suggests that these two proteins do not act cooperatively in enhancing D-loop formation.

Because only a partial depletion of RAD51AP1 can be accomplished by RNA interference, it seems very likely that HR and homology-directed DNA repair are much more dependent on RAD51AP1 than can be revealed in our experiments. The fact that the RAD51-mediated D-loop reaction shows a strong dependence on RAD51AP1 supports this premise. Interestingly, RAD51AP1 expression levels seem to be altered in aggressive lymphoma and other cancers and, similar to RAD51, its expression level is cell cycle regulated (Song et al., 2004; Henson et al., 2006). It will be important to determine whether or not altered expression of RAD51AP1 contributes to the acquisition of the tumor phenotype.

Experimental Procedures

Analyses of Chromosomal Aberrations

HeLa cells were treated with colcemid (100 ng/ml) for 4 h, detached from the culture vessel, washed in PBS and allowed to swell in 75 mM KCl at 37°C for 10 min. Cells were treated with methanol:acetic acid (3:1), dropped on wet slides, air dried, and stained in 3% Giemsa solution in Sørensen phosphate buffer for 10 min. Slides were covered with mounting media (Vectashield 60) and analyzed with a Zeiss Axioskop using a 100× lens with oil and 2000× magnification. We analyzed 50 to 100 metaphases in each case. Gaps were counted as a chromatid break (ctb) when the gap size was wider than the chromatid.

Recombination Assay and Flow Cytometry

The TK6-neo-DRGFP cell line (clone 1-7) has been described elsewhere (Wiese et al., 2002b). I-SceI was expressed transiently from the pCβAsce expression vector (Richardson et al., 1998) and co-transfected with the plasmids encoding the shRNA, as indicated. Forty µg of each plasmid were used to transfect 4 × 106 cells suspended in 650 µl opti-MEM medium (Invitrogen) by electroporation (625 V/cm, 950 µF). Transfected cells were kept in regular growth medium for 24 h and then maintained in regular growth medium containing 100 µg/ml hygromycin B for 2 days. Transfected cells were analyzed by flow cytometry 3 days after electroporation to measure the percentage of cells expressing GFP (Wiese et al., 2002b).

DNA Mobility Shift Assay

RAD51AP1 (0.03 to 1.5 µM) was incubated with 30 nM of ssDNA, dsDNA, or the D-loop substrate in 10 µl of reaction buffer (50 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM MgCl2, 1 mM dithiothreitol and 100 µg/ml BSA) at 37°C for 5 min. The reaction mixtures were resolved in either 5% or 10% polyacrylamide gels in TAE buffer (40 mM Tris acetate, pH 7.4, 0.5 mM EDTA) at 4°C, followed by gel drying and quantification by phosphorimaging analysis. The substrate preparation procedure is described in the Supplemental Section.

D-loop Assay

This was conducted essentially as described (Chi et al., 2006). Briefly, the 32P-labeled 90-mer oligonucleotide substrate (2.4 µM nucleotides) was incubated for 5 min at 37°C with RAD51 (0.8 µM) in 10.5 µl reaction buffer (35 mM Tris, pH 7.5, 1 mM dithiothreitol, 5 mM MgCl2, 50 mM KCl, containing either 2 mM ATP or AMP-PNP). The indicated amount of wild type or mutant RAD51AP1 was then added in 1 µl, followed by a 5-min incubation at 37°C. After the addition of pBluescript replicative form I DNA (35 µM base pairs) in 1 µl, the reaction mixtures were incubated for 6 min at 37°C and then subject to agarose gel electrophoresis. The radiolabeled DNA species were visualized and quantified by phosphorimaging analysis. The same procedure was used to analyze the effect of RAD51AP1 on yRad51 or RAD51 K133R.

Additional Experimental Procedures

The details are provided in the Supplemental Section.

Supplementary Material

01

Acknowledgments

We thank Oleg Kovalenko for material and Hector Nolla for help with flow cytometry. We are grateful to Mauro Modesti and Roland Kanaar for communicating results regarding D-loop binding by RAD51AP1. This work was supported by DOE Low Dose grant 441E, LBNL LDRD grant, NASA grant NNJ05HI36I, Public Health Service grants RO1CA110415, RO1ES015252, and P01CA92584, and Susan G. Komen postdoctoral fellowship PDF0503471.

Footnotes

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