Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases eta and REV1 - PubMed (original) (raw)

Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases eta and REV1

Parie Garg et al. Proc Natl Acad Sci U S A. 2005.

Abstract

In response to DNA damage, the Rad6/Rad18 ubiquitin-conjugating complex monoubiquitinates the replication clamp proliferating cell nuclear antigen (PCNA) at Lys-164. Although ubiquitination of PCNA is recognized as an essential step in initiating postreplication repair, the mechanistic relevance of this modification has remained elusive. Here, we describe a robust in vitro system that ubiquitinates yeast PCNA specifically on Lys-164. Significantly, only those PCNA clamps that are appropriately loaded around effector DNA by its loader, replication factor C, are ubiquitinated. This observation suggests that, in vitro, only PCNA present at stalled replication forks is ubiquitinated. Ubiquitinated PCNA displays the same replicative functions as unmodified PCNA. These functions include loading onto DNA by replication factor C, as well as Okazaki fragment synthesis and maturation by the PCNA-coordinated actions of DNA polymerase delta, the flap endonuclease FEN1, and DNA ligase I. However, whereas the activity of DNA polymerase zeta remains unaffected by ubiquitination of PCNA, ubiquitinated PCNA specifically activates two key enzymes in translesion synthesis: DNA polymerase eta, the yeast Xeroderma pigmentosum ortholog, and Rev1, a deoxycytidyl transferase that functions in organizing the mutagenic DNA replication machinery. We propose that ubiquitination of PCNA increases its functionality as a sliding clamp to promote mutagenic DNA replication.

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Figures

Fig. 1.

Fig. 1.

DNA-bound PCNA is ubiquitinated at Lys-164. (A) Scheme of the assay. (B) Factor requirement for efficient PCNA ubiquitination. (C) Human ubiquitin is linked to PCNA. The standard PCNA ubiquitination assay (described in Materials and Methods) contained either the tagged yeast ubiquitin (His6-Ubi, left lane) or human ubiquitin (right lane). (D) Time course of ubiquitination. Assays were carried out on unprimed or DECAprimed ssDNA. (E) Ubiquitination of mutant PCNAs. Standard assays contained unlabeled PCNA or the indicated mutant PCNA. Detection was by Western blot analysis with rabbit antibodies to PCNA.

Fig. 2.

Fig. 2.

Biochemical properties of PCNAUbi in DNA replication. (A) Purification scheme. Pr-DNA is DECAprimed Bluescript SKII ssDNA. (B) Gel analysis (11% SDS/PAGE) of PCNA and purified PCNAUbi. Staining was with colloidal Coomassie blue. Scanning of the gel showed that ≈3% nonubiquitinated PCNA remained in the purified preparation. (C) Replication of RPA-coated singly primed mp18 ssDNA (60-fmol circles) by Pol δ (120 fmol) and RFC (120 fmol) with increasing PCNA or PCNAUbi was performed essentially as described (38). [α-32P]dTTP was used as radioactive tracer. The PCNA was loaded by RFC for 1 min at 30°C, Pol δ was added, and incubation continued for an additional 90 sec. Products were analyzed on a 1% alkaline agarose gel. (D) Scheme for measuring Okazaki fragment maturation kinetics. (E) Replication and maturation assays of RPA-coated circular ssDNA (100 fmol), primed with an RNA8DNA22 primer, by RFC (200 fmol), PCNA or PCNAUbi (300 fmol), Pol δ (200 fmol), FEN1 (200 fmol), and DNA ligase I (500 fmol) were exactly as described (28). Replication products after the indicated times were separated on a 1% agarose gel in the presence of 0.5 μg/ml ethidium bromide. This method separates covalently closed circular DNA from nicked circular DNA. [α-32P]dTTP was used as radioactive tracer.

Fig. 3.

Fig. 3.

PCNAUbi activates TLS of abasic sites by Pol η.(A) Scheme of substrate V9AP1/C12-4. The gray circles indicate the biotin-streptavidin blocks. The abasic site is shown as 0. (B) Stimulation of bypass by PCNA or PCNAUbi. Standard assays (described in Materials and Methods) were for 5 min with the indicated polymerase and the indicated form of PCNA. The template sequence of interest is shown at left. (C) PCNAUbi stimulates abasic bypass by Pol η. Standard assays with increasing clamp levels were carried out for 60 sec at 30°C. Full-length replication products as the percentage of total primer extension products were quantitated.

Fig. 4.

Fig. 4.

PCNAUbi activates TLS of abasic sites by Rev1. (A) Scheme of substrates V9/C12 and V9AP2/C12. The abasic site is shown as 0.(B) PCNAUbi stimulates Rev1. Standard assays (described in Materials and Methods) contained either the normal template (Upper) or the abasic site template (Lower) and the indicated levels of clamp. The template sequence of interest is shown at left. (C) Control assays for PCNAUbi-mediated Rev1 activity. Either PCNAUbi (lane 1) or RFC (lane 3) was omitted from the standard assay with Rev1 (lane 2). Standard assays without PCNA (lanes 4–6) or with PCNAUbi (lanes 7–9) contained all four dNTPs (4, lanes 4 and 7), only dCTP (c, lanes 5 and 8), or dTTP, dATP, and dGTP (tag, lanes 6 and 9). (D) Time-course assays of PCNAUbi-mediated Rev1 activity at the abasic site. Standard assays were followed with time and insertion opposite the abasic site as percentage of total products was quantitated by using PhosphorImager analysis.

Fig. 5.

Fig. 5.

PCNAUbi activates Rev1 during “standing start” TLS. (A) Scheme of substrate V9AP1/C12. The abasic site is shown as 0.(B) Time course of PCNAUbi-mediated Rev1 activity at an abasic site. Standard assays were used (described in Materials and Methods), and insertion opposite the abasic site (% TLS) was quantitated.

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