PCNA Is Efficiently Loaded on the DNA Recombination Intermediate

Home , Replication factor C, RFC2, RFC3, RFC4, RFC5

PCNA is efﬁciently loaded on the DNA recombination intermediate to modulate polymerase δ, η, and ζ activities

Jian Lia,b, Donald L. Holzschua,b, and Tomohiko Sugiyamaa,b,1

aDepartment of Biological Sciences and bMolecular and Cellular Biology Graduate Program, Ohio University, Athens, OH 45701

Edited by Richard D. Kolodner, Ludwig Institute for Cancer Research, La Jolla, CA, and approved March 27, 2013 (received for review December 22, 2012) Proliferating cell nuclear antigen (PCNA) is required for DNA polymerases Pol η and Pol ζ also contribute to HR. Pol η-deficient homologous recombination (HR), but its exact role is unclear. Here, chicken DT40 cells have defects in gene conversion at the IgG we investigated the loading of PCNA onto a synthetic D-loop (DL) locus (23), and purified human Pol η can catalyze DNA synthesis − − intermediate of HR and the functional interactions of PCNA with within a synthetic DL (24). Loss of Pol ζ function (REV3 / ) Rad51 recombinase and DNA polymerase (Pol) δ, Pol η, and Pol ζ. resulted in increased sensitivity to DNA damaging reagents in PCNA was loaded onto the synthetic DL as efficiently as it was mouse (25) and chicken cells (26, 27). Although Pol ζ is not es- loaded onto a primed DNA substrate. Efficient PCNA loading sential for HR in yeast, the deletion of the yeast REV3 gene greatly requires Replication Protein A, which is associated with the dis- decreases the mutation rate near a DSB because of its low fidelity placed ssDNA loop and provides a binding site for the clamp-loader (28), indicating a role of Pol ζ in HR. Replication Factor C. Loaded PCNA greatly stimulates DNA synthe- Proliferating cell nuclear antigen (PCNA) is a DNA sliding sis by Pol δ within the DL but does not affect primer recognition by clamp, which is conserved in all three domains of life (29). δ Pol . This suggests that the essential role of PCNA in HR is not PCNA is loaded onto DNA by the “clamp-loader” Replication δ δ recruitment of Pol to the DL but stimulation of Pol to displace Factor C (RFC; complex of Rfc1, Rfc2, Rfc3, Rfc4, and Rfc5), η ζ a DNA strand during DL extension. Both Pol and Pol extended which opens and reseals the PCNA on dsDNA in an ATP- fi δ the DL more ef ciently than Pol in the absence of PCNA, but little dependent manner (30–33). Loaded PCNA encircles and slides or no stimulation was observed in the presence of PCNA. Finally, freely along the DNA, anchoring a DNA polymerase to the DNA Rad51 inhibited both the loading of PCNA onto the DL and the for processive DNA synthesis (34–36). In vitro, RFC can load extension of the DL by Pol δ and Pol η. However, preloaded PCNA PCNA onto DNA with 5′ junctions (i.e., junctions between the on the DL counteracts the Rad51-mediated inhibition of the DL ssDNA template and 5′ end of the primer) and nicked dsDNA in extension. This suggests that the inhibition of postinvasion DNA the absence of RPA (37–40). However, in the presence of RPA, synthesis by Rad51 occurs mostly at the step of PCNA loading. PCNA is uniquely loaded to 3′ junctions (i.e., junction between the ssDNA template and the 3′ end of the primer). RPA directly DNA repair | translesion polymerase | sliding clamp interacts with RFC to facilitate the specific binding of RFC to the 3′ junction, thereby directing RFC to load PCNA onto the NA double-strand breaks (DSBs) are introduced into the specific DNA structure (31, 35, 41, 42). Dgenome by several factors, including ionizing radiation, mu- A genetic study in yeast indicated that PCNA is essential for tagenic chemicals, reactive oxygen species, and stalled DNA rep- the postinvasion DNA synthesis during HR (22). Consistently, an lication (1). Without appropriate repair, DSBs may lead to cell in vitro study indicated that PCNA is required for DNA synthesis lethality or cancer (2–4). Homologous recombination (HR) is following Rad51-mediated strand invasion (43). However, sev- a widely conserved essential mechanism for high-fidelity repair of – eral questions remain unanswered regarding the molecular DSBs (5 8). HR is a highly coordinated multistep biochemical fi Sac- functions of PCNA in HR. First, is PCNA loaded ef ciently onto process, which is most elegantly demonstrated in the yeast ′ charomyces cerevisiae. Briefly, DSB ends are first processed by a DL that lacks an ssDNA region beyond the 3 end of the in- ′ – vading strand, which is believed to be crucial for PCNA loading? specialized exonucleases to generate 3 overhangs (9 11), which fi fi are then coated with the ssDNA binding protein Replication Pro- Loading ef ciency of PCNA on a DL has not been quanti ed tein A (RPA). The Rad52 recombination mediator interacts with because the DL is structurally different from most DNA sub- RPA and recruits Rad51 recombinase onto the ssDNA to form strates that have been analyzed for PCNA loading. If it is loaded, a helical nucleoprotein filament (12–15). This Rad51-ssDNA fila- what is the role of RPA in the PCNA loading? Second, is the ment mediates strand invasion into a homologous dsDNA (16) to loaded PCNA oriented in the effective direction? Even if PCNA produce a DNA structure that is referred to as the D-loop (DL; see was loaded on the DL, there are two possible directions of Fig. 6B). The 3′ end of the invading strand is used as the primer loaded PCNA relative to the invading DNA strand. Only one by DNA polymerases for DNA synthesis (postinvasion DNA syn- direction is “productive” in stimulating polymerases (35). The thesis), extending the DL (see Fig. 6F). After postinvasion DNA ratio of the functional PCNA in loaded PCNA on the DL has not synthesis, repair may be completed by a break-induced replication, been quantified. Third, what role does Rad51 recombinase play DSB repair, or synthesis-dependent strand-annealing pathway (5). in the PCNA loading process? To answer these questions, we Postinvasion DNA synthesis is crucial to high-fidelity repair of investigated the loading of PCNA on a synthetic DL in vitro and DSBs because it recovers the genetic information that might be lost during breakage events. Several DNA polymerases, including polymerase (Pol) δ, Pol η, and Pol ζ, are possibly involved in this Author contributions: J.L. and T.S. designed research; J.L., D.L.H., and T.S. performed process. Pol δ is known as a replicative polymerase that constitutes research; J.L. and T.S. analyzed data; and J.L. and T.S. wrote the paper. the replisome (17, 18). Genetic studies in yeast indicate that Pol δ The authors declare no conflict of interest. is involved in mitotic gene conversion (19), meiotic recombination This article is a PNAS Direct Submission. (20), repair of γ-ray–induced DNA damage (21), and homothallic 1To whom correspondence should be addressed. E-mail: [email protected]. switching (HO) endonuclease-induced gene conversion (22). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Studies of HR in vertebrate systems have shown that translesion 1073/pnas.1222241110/-/DCSupplemental.

7672–7677 | PNAS | May 7, 2013 | vol. 110 | no. 19 www.pnas.org/cgi/doi/10.1073/pnas.1222241110 Downloaded by guest on October 1, 2021 the ability of the loaded PCNA to stimulate DNA synthesis by to slide off. These data show that the PCNA is efﬁciently loaded Pol δ, Pol η, and Pol ζ. onto the dsDNA region inside the DL.

Results RPA on the ssDNA Loop Stimulates PCNA Loading. Previous studies PCNA Is Loaded on the DL and Primed ssDNA with Similar Efficiency. using primed ssDNA substrates indicated that efficient PCNA We adopted the method published by Podust et al. (44) for loading required an ssDNA region beyond the 3′ junction of the quantitative analyses of PCNA loading onto DNA substrates with primer and template (37, 38). This ssDNA region is believed to be various structures. Essentially, we constructed 32P-labeled PCNA complexed with RPA, which provides a binding site for RFC for (32P-PCNA) and tested its loading onto unlabeled DNA molecules subsequent PCNA loading. However, our DL substrate had only in vitro (details are provided in SI Materials and Methods). In a one-nucleotide ssDNA region at the 3′ end of the invading a standard reaction, the preannealed DNA substrate was first in- DNA strand, which is too small for RPA binding (48, 49) yet cubated with RPA, followed by RFC and 32P-PCNA. Three required RPA for PCNA loading. Therefore, we performed a minutes after the addition of PCNA, the reaction mixture was series of experiments to identify the location of RPA in PCNA E H treated with glutaraldehyde to fix the PCNA ring structure in its loading using different DNA structures (Fig. 1 and ). In- closed conformation on the DNA substrate and subjected to aga- terestingly, the loading of PCNA on the DL substrates containing A fi fi 3′ junctions [DL-left (L) and DL2-L] was not better than that rose gel electrophoresis (Fig. 1 ). We rst con rmed the loading of ′ 32P-PCNA on a primed Bluescript ssDNA substrate (Fig. S1). As on the DL substrates containing 5 junctions [DL-right (R) and – DL2-R]. That was also confirmed by analyzing the PCNA- expected, a low-mobility PCNA DNA complex was produced in δ the presence of RFC and ATP. The reaction was less efficient with dependent extension of the DLs by Pol (Fig. S3), where DL, DL2-L, and DL2-R gave comparable amounts of full-length unprimed ssDNA or dsDNA (Fig. S1C), as previously reported (45, products, indicating that PCNA was loaded onto these substrates 46). We then analyzed the loading of the 32P-PCNA on a synthetic with similar efficiency. All these data indicate that PCNA loading substrate that mimicked the DL and on a primed ssDNA substrate on the DL was not dependent on a 3′ junction, in contrast to (C2) (Fig. 1B). When we used the C2 substrate (Fig. 1C,lanes1–4), the previous studies using primed ssDNA substrates (30–33). 32P-PCNA mobility was shifted in the gel in the presence of RPA, C Therefore, RPA most likely binds to the displaced ssDNA loop RFC, and ATP (Fig. 1 , lane 4), indicating that it was loaded on to stimulate the PCNA loading reaction. To investigate this hy- C2. Although C2 is a linear DNA molecule, loaded PCNA did not pothesis, we analyzed PCNA loading on a modified DL substrate, slide off from the substrate because both ends of the DNA were where the ssDNA loop was annealed to another oligonucleotide complexed with RPA, as previously reported (42, 47). Likewise, to make it double-stranded (Fig. 2A, DL-CAP). The loading of C – when the DL substrate was used (Fig. 1 ,lanes58), a mobility 32P-PCNA on the DL-CAP was greatly reduced, supporting the C shift was observed in the complete reaction (Fig. 1 , lane 8) but not notion that RPA binding to the displaced ssDNA region of the C – in the absence of RFC, ATP, or RPA (Fig. 1 ,lanes5 7) indicating DL is necessary for PCNA loading. that PCNA was loaded on the DL substrate. PCNA loading in the Because studies have shown that RPA/RFC protein–protein complete reactions on the C2 and DL substrates was comparable interaction is involved in the PCNA loading (31, 41, 42) on (Fig. 1F). The kinetics of loading on DL and C2 were similar (Fig. simple primed substrates, we investigated whether this is also the 1G), and loading was dependent on the amount of RFC supplied case in the PCNA loading on the DL. To do this, we replaced (Fig. S2). To identify the region of the DL where PCNA was RPA with Escherichia coli single strand binding (SSB) protein in loaded, we treated the product of the complete loading reaction our loading protocol (Fig. 2 B and C). SSB only poorly stimu- with KpnI to cleave the dsDNA region in the DL (Fig. 1B). Five lated the loading of PCNA on the DL (Fig. 2B, lane 5) and C2 minutes of KpnI digestion did not release the loaded PCNA from (Fig. 2B, lane 2). Additionally, RPA titration indicated that the the substrate; however, after 30 min of incubation, most of the amount of RPA required for PCNA loading roughly corresponded 32P-PCNA was released from the complex (Fig. 1D), indicating to that required for full occupancy of the displaced ssDNA loop that KpnI digestion opens the DL and allows the loaded PCNA (Fig. S4 C and D). Based on these results, we conclude that RPA BIOCHEMISTRY

Fig. 1. PCNA is efﬁciently loaded on the DL. (A) Illustration of the PCNA loading assay. A detailed description is provided in SI Materials and Methods. (B) Structures of synthetic DNA substrates (DL and C2) used for the PCNA loading analysis. Numbers are lengths of DNA segments (in nucleotides). (C) Loading reactions of 32P-PCNA on C2 and DL were performed in the presence or absence of ATP, RFC, and RPA as indicated. (D) Complete PCNA loading reaction with DL substrate was followed by incubation with (lanes 2 and 4) or without (lanes 1 and 3) KpnI for indicated times. (E) PCNA loading reactions were carried out using DNA substrates with different structures (DL, DL-L, DL-R, DL2-L, and DL2-R), which are illustrated in H.(F and H) Percentages of loaded PCNA were calculated from C and E, respectively, with repeated experiments (n = 3). (G) Same reactions as shown in lane 4 (C2) and lane 8 (DL) of C were stopped at the indicated time points. Error bars represent the SDs.

Li et al. PNAS | May 7, 2013 | vol. 110 | no. 19 | 7673 Downloaded by guest on October 1, 2021 almost no loading was observed. In contrast, when Rad51 was added to the DL after the PCNA loading reaction, a supershift of the signal was observed (Fig. 3B), indicating the formation of a Rad51–PCNA–DNA complex. No significant effect on the percentage of the loaded PCNA was observed (Fig. 3D, PCNA→Rad51). These results indicate that Rad51 inhibits the loading of PCNA but does not affect the stability of the loaded PCNA. Because the reaction contained RPA that should occupy the ssDNA loop, Rad51 should preferentially bind to the dsDNA Fig. 2. RPA on the ssDNA loop stimulates PCNA loading on the DL. (A) PCNA region of the DL (Fig. 3A). This was confirmed by measuring the loading was performed on the DL (lanes 3 and 4) and its derivative that had ATPase activity of Rad51 under the same conditions (Fig. S5). an additional oligonucleotide covering the ssDNA loop (DL-CAP; lanes 1 and Therefore, the Rad51-mediated inhibition of PCNA loading was – – 2). (B) PCNA loading on the C2 (lanes 1 3) and the DL (lanes 4 6) was per- not due to the removal of RPA from the DL or the melting of the formed under standard conditions in the presence or absence of RFC (0.08 – μM), RPA (0.8 μM), and E. coli SSB protein (0.8 μM) as indicated. (C) Per- DL structure into ssDNA but to the Rad51 dsDNA complex in centages of loaded PCNA were calculated from B with repeated experiments the DL. Most likely, Rad51 bound to the invading DNA strand (n = 3–4). Error bars represent the SDs. acts as a physical obstruction preventing the loading of PCNA. Unexpectedly, even when Rad51 and ssDNA were preincubated before reconstituting the Rad51–DL complex, maximum inhibition on the ssDNA loop provides the binding site for the RFC, leading required Rad51 that could saturate the entire dsDNA region of the to PCNA loading onto the dsDNA region within the DL. DL (Rad51/DL ratio = 32; Fig. 3 C and D). This might suggest that Rad51 molecules outside the invading DNA strand may also con- Rad51 Inhibits PCNA Loading on the DL. Although Rad51-dependent tribute to the inhibition. Alternatively, Rad51 on the invading DNA strand invasion is crucial for HR, an increasing number of DNA strand may quickly redistribute along the dsDNA region after studies have indicated that Rad51 inhibits the process after DNA DNA strand invasion. Consistent with the latter idea, the Rad51- strand invasion (50–52). Therefore, it is of special interest to in- ssDNA preincubation inhibited the PCNA loading more strongly vestigate the effect of Rad51 in PCNA loading on the DL (Fig. 3). during first 2 min of the loading reaction (Fig. S6, Left;compare When the RPA–DL complex was incubated with an increasing lower two curves). amount of Rad51 and then subjected to the PCNA loading re- δ action, the loading of 32P-PCNA was inhibited by Rad51 in a PCNA Stimulates Pol to Displace DNA Strand During the Postinvasion concentration-dependent manner (Fig. 3 A and D, red open cir- DNA Synthesis. Genetic and biochemical studies in yeast indicated fi that both PCNA and Pol δ play major roles in HR (19–22). cle). At the concentration of Rad51 that was suf cient to saturate η ζ all dsDNA regions of the DL substrate (Rad51/DNA = 32), Studies in vertebrate systems showed that Pol and Pol are also involved in HR (23, 26). To characterize the roles of these polymerases in HR further, we investigated the ability of Pol δ (Pol3–Pol31–Pol32 complex), Pol η (Rad30), and Pol ζ (Rev3– Rev7 complex) to extend the DL in the presence and absence of loaded PCNA (Fig. 4). To distinguish primer recognition and strand displacement in DL extension, we used the DL substrate that had a one-nucleotide ssDNA region beyond the 3′ end of the primer (Fig. 1B). The polymerase can extend the primer by 1 nt before encountering the dsDNA region of the DL substrate. Among the three yeast polymerases we tested, Pol δ showed the poorest ability to extend the DL in the absence of the loaded PCNA [Fig. 4 C (compare lanes 2, 4, and 6) and D], consistent with the previous observations (24, 53). Interestingly, however, Pol δ extended almost all the primers for 1–2 nt (Fig. 4C, lane 4), indicating that Pol δ efficiently recognized the primer in the DL in the absence of PCNA but that the synthesis was obstructed by dsDNA. When Pol δ was added to the DL that had loaded PCNA (Fig. 4 C and D, lanes 5 and 11–13, respectively), the majority of primers were fully extended, indicating that PCNA greatly stimulated Pol δ’s ability to displace the DNA strand during DNA synthesis. Both the inability of Pol δ to extend the DL and the stimulation by PCNA were also reproduced with the plasmid-sized substrate with a 457-nt loop [Fig. 4 B (“Bubble” “ ” F Fig. 3. Rad51 inhibits PCNA loading. (A) Rad51 (0, 0.4, 0.8, 1.6, 2.4, and 3.2 μM and Y-shape ) and (lanes 7, 8, and 11)], indicating that these from lanes 2–7) was added to 0.1 μM preannealed DL preincubated with activities are unrelated to the loop size. 0.4 μM RPA. 32P-PCNA and RFC were then added to start the PCNA loading reaction. (B) Same experiment as in A was performed, except that Rad51 was Pol δ, Pol η, and Pol ζ Respond Differently to PCNA on the DL. When added after PCNA loading on the DL. (C) Rad51–ssDNA complex was first yeast Pol η was added, the DL was extended efficiently without produced by incubating Rad51 (0, 0.4, 0.8, 1.6, 2.4, and 3.2 μM from lanes 2–7) the loaded PCNA (Fig. 4 C and D, lanes 2 and 2–4, respectively), with 0.1 μM 29-mer oligo (TSO319), which mimicked invading strand for 5 min consistent with the previous observations (24, 43, 53). This ex- at 37 °C. The reaction mixture was further incubated with preannealed DL tension was not significantly affected by loaded PCNA (Fig. 4 C (0.1 μM) without the 29-mer for 15 min and then with 0.4 μM RPA for another 32 and D, lanes 3 and 5–7, respectively). However, on the plasmid- 3 min at 37 °C. P-PCNA and RFC were then added to start the PCNA loading sized DL, extension by Pol η was moderately but clearly stimu- reaction. (D) Experiments shown in A (red open circle), B (blue square), and C F (black triangle) were repeated, and percentages of PCNA that were in the lated by the loaded PCNA (Fig. 4 , lanes 3 and 4). This may complexes were calculated and plotted against the Rad51/DL molecular ratio. suggest that Pol η has lower efficiency to interact with PCNA but Error bars represent the SDs (n = 3). that once they interact, Pol η-PCNA can extend longer than 100

7674 | www.pnas.org/cgi/doi/10.1073/pnas.1222241110 Li et al. Downloaded by guest on October 1, 2021 Fig. 4. Extension of the DL by Pol δ, Pol η, and Pol ζ is differently modulated by PCNA. (A) Illustration of DNA polymerase assay on the DL containing a 32P-labeled invading DNA strand (red arrow). Nonradiolabeled PCNA was loaded on the DL, and Pol δ, Pol η, or Pol ζ was then added to start DNA synthesis. (B) Plasmid-sized substrates that were used in F are shown. A 32P-labeled primer (40-mer) was annealed to 3.0 kb of dsDNA containing a 457-nt non- complementary region (Bubble), which was produced from Bluescript SK(+) and SK(−) ssDNA molecules (details are provided in SI Materials and Methods). + The same primer was annealed to a Y-shaped derivative (Y-shape) and linearized Bluescript SK ssDNA (Linear). (C) DL substrate was incubated with PCNA and RFC for 3 min as indicated, and then with Pol η, Pol ζ, or Pol δ for 15 min. DNA products were analyzed by denaturing PAGE. (D) Time courses of the DNA synthesis in DL substrate by indicated polymerases were analyzed in the presence or absence of PCNA/RFC. (E) Same reactions as in D were repeated, except that simple primed substrate (C2; Fig. 1B) was used instead of DL. (F) Plasmid-sized DNA substrates shown in B were used for the PCNA loading and DNA synthesis reactions by Pol δ,Polη, Pol ζ, or Klenow fragment (K). Lanes 1, 10, and 13 contain size markers that were produced by labeling a 100-bp DNA ladder by T4-kinase followed by denaturing in formamide.

nt. In control reactions (Fig. 4E), both Pol η and Pol δ extended extension by polymerases. However, it was unclear whether Rad51 the primer on simple primed substrate both in the presence and would inhibit the DL extension if PCNA had been loaded already absence of the loaded PCNA. Pol ζ extended the simple primed (Fig. 5A). To address this question, we examined the effect of substrate less efficiently than the same amount of Pol η and Pol δ Rad51 on DNA synthesis by Pol δ and Pol η in the DL in the (Fig. 4E). Interestingly, Pol ζ extended the DL substrate slightly presence or the absence of loaded PCNA (Fig. 5). As expected, more efficiently than Pol δ in the absence of PCNA (Fig. 4 C and in the absence of PCNA, Rad51 strongly inhibited both Pol δ and BIOCHEMISTRY D, lanes 6 and 14–16, respectively), indicating that Pol ζ exten- Pol η extension of the DL substrate [Fig. 5 B and C (lanes 2–8) sion was not strongly inhibited by the DL structure. However, no and D and E (blue curves)]. Importantly, if PCNA was preloaded, stimulation was observed in the presence of PCNA (Fig. 4 C, D, Rad51 inhibition of DNA synthesis by Pol η [Fig. 5 B (lanes 9–15) and F; Pol ζ). and D] and Pol δ [Fig. 5 C (lanes 9–15) and E] was less pro- We detected an unexpected premature termination product in nounced. These results indicate that loaded PCNA lessens the the Pol δ extension reactions on the simple primed substrate C2 inhibition by Rad51. (indicated by the asterisk in Fig. 4E). It was not detected on the DL that had the same sequence as C2 (Fig. 4D) or on other Discussion primed DNA substrates (Fig. S3, substrate C3). We think that this In this study, we describe unique findings about the functional sequence-specific termination is due to the secondary structure of interactions of RFC, PCNA, DNA polymerases, and Rad51 the C2 substrate. We also observed a small fraction of unused recombinase on the DNA recombination intermediate. Based on primers in the presence of PCNA and RFC (Fig. 4 C–E). This our results, we propose the updated DSB repair model, focusing apparent inhibition of primer recognition was observed consis- on the events after Rad51-mediated strand invasion (Fig. 6). tently in the reactions containing RFC and was independent of First, we showed that PCNA loading on the DL structure was PCNA or polymerase type. Because the reaction contained very efficient (Fig. 1). In addition, PCNA loading onto the DL fourfold more RFC than DNA substrate, the excess RFC on the required the binding of RPA to the displaced ssDNA loop, DNA might inhibit primer recognition by polymerases. We do not thereby providing a binding site for RFC, which mediated the believe that this inhibition is biologically significant. PCNA loading (Fig. 2). Loaded PCNA greatly stimulated Pol δ extension within the DL (Fig. 4). Without PCNA, Pol δ was very Loaded PCNA Counteracts the Rad51-Mediated Inhibition of DNA poor at extending the DL. The DL structure itself did not in- Synthesis. A previous study has shown that on the invading DNA terfere with the polymerase–primer interaction. Therefore, the strand, Rad51 inhibits DNA synthesis (54). We have shown here major role of PCNA in the DL extension is to stimulate Pol δ to that Rad51 inhibits the loading of PCNA on the DL. Therefore, we displace the DNA strand. In contrast, Pol η extended the DL expected that Rad51 would also inhibit PCNA-dependent DL much more efficiently than Pol δ in the absence of PCNA, but

Li et al. PNAS | May 7, 2013 | vol. 110 | no. 19 | 7675 Downloaded by guest on October 1, 2021 simple primed ssDNA substrates showed that the 3′-oriented loading of PCNA depends on RPA, which directionally binds to the ssDNA template (29, 38). However, in the DL analyzed in this study, RPA binds to the displaced ssDNA, which is running in the opposite direction to the template strand. Precise molecular ar- chitecture of the RPA–RFC–PCNA complex on the DL needs to be demonstrated to reconcile this apparent inconsistency. A previous study indicated that Rad51 recombinase inhibited the primer extension by means of the Klenow enzyme (54). We found that Rad51 inhibited both PCNA loading on the DL (Fig. 3) and DNA synthesis (Fig. 5). Therefore, Rad51 can inhibit two essential biochemical processes of the postinvasion DNA synthesis. It is not clear which of these two inhibitions is more im- portant in regulating HR. However, it seems more likely that PCNA loading is a key regulatory target of Rad51, because PCNA loading precedes the Pol δ–primer interaction (18). Two pieces of data in this paper are consistent with this idea. First, in Rad51 titration experiments (Figs. 3D and 5D and E), the Rad51 concentration needed to inhibit PCNA loading was lower than that required to inhibit primer extension, indicating that Rad51 is a more effective inhibitor of PCNA loading than of DNA synthesis per se. Second, when PCNA was preloaded on the DL, Rad51 allowed polymerase to extend the DL more easily (Fig. 5). Although we do not know whether PCNA can be loaded before removing Rad51 in vivo, this in vitro result suggests that the inhibition of PCNA loading is more crucial for regulation of postinvasion DNA synthesis. These Rad51-mediated inhibitions

Fig. 5. Preloaded PCNA counteracts Rad51 inhibitory effect on Pol δ and Pol η.(A) Rad51 was added to DL preloaded with PCNA. DNA synthesis was then started by means of the addition of Pol δ or Pol η.(B and C) Rad51 (0, 0.2, 0.4, 0.6, 0.8, 1.2, and 1.6 μM) was added to 0.02 μM DL (lanes 2–8) or DL preloaded with PCNA (lanes 9–15). Then, DNA synthesis was started by adding 0.02 μM Pol η (B) or Pol δ (C). (D and E) Quantiﬁcation of the relative percentage of primer use in B and C, respectively. The values were nor- malized by setting the value of no Rad51 reaction to be 100%. Error bars represent SDs (n = 3).

most of the products were shorter than 100 nt. PCNA stimulated the reaction to extend a much longer distance. Taken together, these results suggest that Pol δ and Pol η may have different roles in HR, dependent on the availability of PCNA (Fig. 6 D and E). In our experiments, Pol ζ demonstrated limited ability to extend DL that was not stimulated by PCNA. However, our Pol ζ preparation did not include the Rev1 subunit previously sug- gested to have a critical role in Pol ζ function in HR (55). Re- cently, three groups reported that Pol31 and Pol32, which were considered subunits of Pol δ, were essential subunits of functional Pol ζ (56–58). The potential roles of these proteins in DL extension by Pol ζ remain to be elucidated. It is known that only one side of the PCNA ring can interact with polymerases (29, 35). Therefore, if the PCNA loading is oriented randomly, only half of the loaded PCNA can stimulate Fig. 6. Model of PCNA functions in HR. (A) A DSB is processed and com- polymerase. As shown in Fig. 4D, the majority of DL substrate plexed with Rad51. (B) DNA strand invasion by Rad51 requires RPA, which produced the full-length product in the presence of PCNA. Im- occupies the ssDNA loop. (C) After mediating the strand invasion, Rad51 portantly, the reaction contained the same amounts (0.2 pmol) of needs to be removed by Rad54. (D) RPA remaining on the ssDNA recruits RFC, which mediates the loading of PCNA onto the invading DNA strand. (F) DNA substrate and PCNA, indicating that the majority of PCNA Loaded PCNA stimulates the activity of Pol δ and Pol η to carry out the in the reaction was loaded on the DL in the correct orientation postinvasion DNA synthesis. (E) In the absence of PCNA, Pol η may extend (oriented to the 3′ end of the primer). Previous studies using the the DNA at lower efﬁciency.

7676 | www.pnas.org/cgi/doi/10.1073/pnas.1222241110 Li et al. Downloaded by guest on October 1, 2021 can be cleared up by Rad54, which removes Rad51 from dsDNA shown in Table S1. Oligonucleotides used to produce each substrate are (54, 59) (Fig. 6C). Rad54-mediated stimulation of PCNA shown in Table S2. loading is yet to be demonstrated in vivo and in vitro. This study contributes to our understanding of the mechanisms of ACKNOWLEDGMENTS. We thank Dr. John Kuriyan (University of California, Berkeley, CA) for providing the PCNA and RFC expression plasmids. We HR-mediated DNA repair and maintenance of genome integrity. thank Wolf Heyer (University of California, Davis, CA) and Noriko Kantake and Sarah Wyss (both from Ohio University) for comments on this manuscript. Materials and Methods This work was partially supported by Ohio University Student Enhancement Details of plasmid construction, protein puriﬁcation, and assays are provided Awards (to J.L.) and by Ohio University Startup Funds and Ohio University in SI Materials and Methods. Sequences of synthetic oligonucleotides are Research Committee Funds (to T.S.).

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