PCNA Is Efficiently Loaded on the DNA Recombination Intermediate To

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 September 28, 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.

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