Figure 3. DNA-bound PCNA bypasses the requirement of a gap for strand-specific MMR in NPE.

(A) Schematic diagram of the assay. Singly biotinylated pMM2^AC (a pMM1 derivative carrying only one BbvCI site) carrying a nick was bound to Sepharose beads and incubated with recombinant hPCNA and hRFC. The nick was then ligated, and the complex was washed with a buffer containing 1 M KCl. The hPCNA-DNA complex was incubated in NPE to test whether MMR occurs. (B) In vitro PCNA-loading reaction. Untreated DNA (top), linearized DNA (middle, by XmnI), and quantitative immunoblottings for hPCNA and hRfc2 (bottom) of samples from the in vitro PCNA-loading assay using covalently closed, and A-strand-nick-carrying pMM2^AC are presented. (*) indicates linear DNA, which was excluded from the calculation of %closed. (C) The hPCNA-DNA complexes described in (B) were split into two portions to test whether PCNA encircles DNA (C) and whether MMR occurs upon incubation in NPE (D). The one portion was treated with either control buffer, or buffer containing XmnI whose cleavage site is located 1382 bp away from the PCNA entry point. DNA from the reaction (top), linearized DNA (middle, by XmnI), and a hPCNA immunoblot (bottom) are shown. Since nick-carrying molecules were accumulated during incubation, the level of closed-circular molecules was lower than the original substrates shown in (B). (D) The other potion of the hPCNA-DNA complexes described in (B) was incubated in NPE. The MMR efficiencies were calculated as described in Figure 1C.

DOI: http://dx.doi.org/10.7554/eLife.15155.011

Figure 3—figure supplement 1. Requirement of PCNA for gap-filling and nick-ligation reactions in Xenopus egg extracts.

(A) Characterization of xPCNA sera. 0.1 μl of NPE and 10 ng of recombinant xPCNA were separated using 4–15% SDS-PAGE and transferred onto PVDF membranes. Each membrane strip was probed with either the xPCNA or pre-immune serum (PI) from the same rabbit. The same exposure sets of PI and xPCNA antibodies are shown. xPCNA (M_r = 2.88 × 10⁴ as a monomer) was detected as nearly a single band in NPE. (*) indicates cross-reacting band. (B) The efficiency of PCNA depletion from HSS. HSS was depleted with pre-immune (lanes 1–4) or xPCNA antibodies (lanes 5–9), and supplemented with either control buffer (lanes 4 and 8) or 0.5 μM recombinant xPCNA (lane 9). HSS (0.5 μl) from each depletion rounds was separated by SDS-PAGE and probed with the indicated antibodies. Orc2 served as a loading control. (C) The gap-filling reaction in PCNA-depleted HSS. Gap-carrying homoduplex pMM1^AT plasmids were incubated in PCNA-depleted HSS described in (B) and sampled at the indicated times. The efficiency of the gap-filling reaction was analyzed using ethidium bromide containing agarose gel electrophoresis. Depletion of PCNA inhibited appearance of closed-form plasmids even after 60 min (lane 12), indicating that PCNA is essential for either DNA synthesis at the gap site, flap-processing, or ligation at the gap in HSS. (*) indicates linearized fragments produced by the nicking endonuclease. (D) The gap-filling reaction in p21-peptide supplemented NPE. Because PCNA-depletion from NPE was not possible due to its high concentration, p21 peptides (Mattock et al. 2001) were used to inhibit PCNA function in NPE. Gap-carrying homoduplex pMM1^AT plasmids were incubated in NPE supplemented with either control buffer (lanes 2–4), 1 mg/ml wildtype (lanes 5–7), PIP mutant (lanes 8–10) or jumbled p21 peptide (lanes 11–13), and sampled at the indicated times. The efficiency of gap filling was analyzed using ethidium bromide containing agarose gel electrophoresis. The wildtype but not the PIP mutant nor jumbled p21 peptide inhibited appearance of closed-form plasmids, suggesting that PCNA is required for either DNA synthesis at the gap site, flap-processing, or ligation at the gap in NPE. (*) indicates linearized fragments produced by the nicking endonuclease. (E) The depletion efficiency of PCNA from HSS. HSS was depleted using pre-immune (lane 1) or xPCNA antibodies (lane 2). Immunoblots of the HSS samples (0.5 μl each) are shown. Orc2 served as a loading control. (*) indicates cross-reacting band. (F) Comparison of nick ligation and gap filling in PCNA-depleted HSS. Nick-carrying homoduplex pMM2^AT (lanes 1–11) or gap-carrying homoduplex pMM1^AT (lanes 12–22) was incubated in either mock-treated or PCNA-depleted HSS described in (E), and sampled at the indicated times. The efficiencies of accumulation of closed-circular molecules were analyzed using ethidium bromide containing agarose gel electrophoresis. Although gap filling was completely inhibited by PCNA-depletion, nick ligation was only partially inhibited, indicating that PCNA is only partially required for nick ligation in HSS.

DOI: http://dx.doi.org/10.7554/eLife.15155.012

Figure 3—figure supplement 2. Characterization of recombinant hRFC and hPCNA.

(A) 300 ng of recombinant hRfc1-5 purified from baculovirus-infected High Five insect cells was separated using SDS-PAGE and stained with Coomassie brilliant blue R-250. (B) 1 μg each of recombinant xPCNA and hPCNA purified from E. coli was separated using SDS-PAGE and stained with Coomassie brilliant blue R-250. (C) The depletion efficiency of PCNA from HSS. HSS was depleted using pre-immune (lane 1) or xPCNA antibodies (lane 2). The HSS samples were separated using SDS-PAGE and probed with the indicated antibodies. Orc2 served as a loading control. (D) hPCNA can replace xPCNA in the MMR reaction in HSS. A-strand-gap-carrying pMM1^AC plasmids were incubated in HSS described in (C) supplemented with either control buffer (lanes 1–6), 0.5 μM of xPCNA (lanes 7–9) or hPCNA (lanes 10–12) and sampled at the indicated times. %repair was calculated as described in Figure 1C. hPCNA, as well as xPCNA, restored the A to G MMR in xPCNA-depleted HSS, indicating that hPCNA can functionally replace xPCNA in MMR. Note that the overall MMR efficiency was weakened because of the rather harsh xPCNA depletion condition (4 round depletion).

DOI: http://dx.doi.org/10.7554/eLife.15155.013