Ubiquitinated Proliferating Cell Nuclear Antigen Activates Translesion DNA Polymerases and REV1
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Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases and REV1 Parie Garg and Peter M. Burgers* Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid, St. Louis, MO 63110 Edited by Jerard Hurwitz, Memorial Sloan–Kettering Cancer Center, New York, NY, and approved November 4, 2005 (received for review July 14, 2005) In response to DNA damage, the Rad6͞Rad18 ubiquitin-conjugat- and requiring additional activation by the Cdc7͞Dbf4 protein ing complex monoubiquitinates the replication clamp proliferating kinase that normally functions in cell cycle progression (5, 12). cell nuclear antigen (PCNA) at Lys-164. Although ubiquitination of It is this complex pathway that mainly contributes to DNA PCNA is recognized as an essential step in initiating postreplication damage induced mutagenesis in eukaryotic cells. Although repair, the mechanistic relevance of this modification has remained normally involved in lagging strand DNA replication, the high- elusive. Here, we describe a robust in vitro system that ubiquiti- fidelity Pol ␦ is also required for damage-induced mutagenesis. nates yeast PCNA specifically on Lys-164. Significantly, only those The functions of Pol and Rev1 appear to be uniquely confined PCNA clamps that are appropriately loaded around effector DNA to mutagenesis. Pol is an error-prone DNA polymerase that can by its loader, replication factor C, are ubiquitinated. This observa- bypass damage (13). Rev1 is a deoxycytidyl transferase that tion suggests that, in vitro, only PCNA present at stalled replication shows the highest catalytic activity opposite template guanines forks is ubiquitinated. Ubiquitinated PCNA displays the same and abasic sites (14, 15). Rev1 is primarily responsible for replicative functions as unmodified PCNA. These functions include inserting dC residues opposite abasic sites during mutagenesis loading onto DNA by replication factor C, as well as Okazaki (16–18). Several models have been proposed in which one DNA fragment synthesis and maturation by the PCNA-coordinated ac- polymerase carries out the insertion step across the lesion, tions of DNA polymerase ␦, the flap endonuclease FEN1, and DNA whereas a second polymerase extends from the lesion (19, 20). ligase I. However, whereas the activity of DNA polymerase Although it is clear that ubiquitination of PCNA is an initi- remains unaffected by ubiquitination of PCNA, ubiquitinated PCNA ating step in postreplication DNA repair, the precise function of specifically activates two key enzymes in translesion synthesis: ubiquitinated PCNA (PCNAUbi) has remained obscure. Several DNA polymerase , the yeast Xeroderma pigmentosum ortholog, possible models for PCNAUbi can be envisaged. PCNAUbi may and Rev1, a deoxycytidyl transferase that functions in organizing function in fork remodeling during preparation for TLS, e.g., it the mutagenic DNA replication machinery. We propose that ubiq- may inhibit further progression of the replicative DNA poly- uitination of PCNA increases its functionality as a sliding clamp to merases. Secondly, PCNAUbi may recruit translesion DNA poly- promote mutagenic DNA replication. merases to sites of DNA damage. Additionally, PCNAUbi could function as a specialized processivity factor for translesion DNA DNA replication ͉ postreplication repair ͉ translesion synthesis ͉ polymerases. Currently, these models have remained unexam- BIOCHEMISTRY ubiquitination ͉ yeast ined for lack of a biochemical source of PCNAUbi. Studies in human cells show colocalization of PCNAUbi with odification of the replication clamp proliferating cell Pol at DNA damage foci that are consistent with, but do not Mnuclear antigen (PCNA) has recently emerged as an prove, a model in which PCNAUbi recruits Pol to sites of DNA important regulatory switch during DNA replication and DNA damage (21, 22). Furthermore, although ubiquitination of damage response. The critical site of modification on PCNA is PCNA is an essential step in mutagenesis, there is no evidence Lys-164, and either sumoylation or ubiquitination at this residue that PCNAUbi can actually function as a processivity factor for can occur (1, 2). Although sumoylation at Lys-164 is proposed to DNA polymerases in a manner similar to PCNA. The simple be associated with normal replicative functions, monoubiquiti- prediction would be that if PCNAUbi would retain this ability, it nation of this residue by the Rad6͞Rad18 ubiquitin E2͞E3 would function specifically in a complex with one or more complex channels DNA damage into the postreplication DNA translesion DNA polymerases (5). However, several studies have repair (PRR) pathway (1, 3, 4). The PRR pathway is comprised already demonstrated that efficient in vitro TLS of damaged of two error-prone branches involving translesion synthesis DNA can be accomplished by a Y-class DNA polymerase in a (TLS) by error-prone DNA polymerases, and an error-free complex with unmodified PCNA. These PCNA-stimulated er- damage avoidance branch that proceeds by fork regression and ror-prone polymerases include yeast and human Pol , and template switching (5, 6). The latter branch depends on further human Pol and Pol (23–26). In addition, the mutagenic Pol modification of monoubiquitinated PCNA involving Rad5 and is also stimulated by PCNA (27). Therefore, data indicating Mms2͞Ubc13 E2͞E3 catalyzed formation of polyubiquitin that a TLS polymerase functions specifically with PCNAUbi chains via an unusual Lys-63 ubiquitin linkage (1). remain unclear. Replicative DNA polymerases of the B-family possess a very To determine the biochemical function of PCNAUbi, we have restrictive active site, which promotes high-fidelity DNA repli- developed a robust assay for in vitro PCNA ubiquitination and cation but inhibits bypass synthesis of many DNA lesions (5, 7, have prepared and purified PCNAUbi on a scale permitting 8). On the other hand, Y-family DNA polymerases are partic- biochemical analysis. We report that ubiquitination does not ularly adapted to the bypass of DNA lesions because of a more appear to affect normal replicative functions of the replication open active site. For instance, DNA polymerase (Pol ), the ortholog of the human Xeroderma pigmentosum protein, can accommodate a cis-syn pyrimidine dimer in its active site Conflict of interest statement: No conflicts declared. allowing facile damage bypass (9). TLS in yeast comprises a This paper was submitted directly (Track II) to the PNAS office. bifurcated pathway in which cis-syn pyrimidine dimers are Abbreviations: PCNA, proliferating cell nuclear antigen; PCNAUbi, ubiquitinated PCNA; primarily bypassed by Pol (10, 11). Most other forms of DNA RFC, replication factor C; RPA, replication protein A; TLS, translesion synthesis. damage are bypassed by a more complex pathway involving the *To whom correspondence should be addressed. E-mail: [email protected]. participation of three DNA polymerases, Pol ␦, Pol , and Rev1, © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505949102 PNAS ͉ December 20, 2005 ͉ vol. 102 ͉ no. 51 ͉ 18361–18366 Downloaded by guest on September 30, 2021 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. clamp. However, surprisingly, PCNAUbi is a specific activator for except that 1.6 mM ATP was used, and the incubation was for two DNA polymerases, Pol and Rev1, that function in the two 45 min. At that time, Ϸ60–90% of the PCNA monomers were defined branches of TLS. ubiquitinated by SDS͞PAGE analysis (three preparations). Ubi- quitinated intermediates of Uba1 and Rad6͞Rad18 were dis- Materials and Methods charged with 10 mM DTT for 10 min at 30°C, and the reaction DNA Substrates. Bluescript SKIIϩ ssDNA, prepared from Esch- was diluted 2-fold and adjusted to 250 mM NaCl. PCNAUbi was erichia coli by using phagemid technology, was primed with batch-bound to a 1-ml Ni͞agarose column in buffer A (25 mM ten 30-mer primers, roughly equally spaced along the circle Hepes⅐NaOH, pH 7.6͞250 mM NaCl) containing 5 mM imida- (DECAprimed DNA). Bluescript ssDNA primed with a single zole. The column was washed with 5 ml of this buffer and RNA–DNA primer was used in the Okazaki fragment matura- step-eluted with buffer A containing 250 mM imidazole. The tion assays (28). mp18 ssDNA primed with a single primer was eluate was diluted with 1.5 vol of buffer B (30 mM Hepes⅐NaOH, used in the processivity assays (29). All oligonucleotide sub- pH 7.6͞1 mM EDTA͞1mMDTT͞0.02% E10C12 (a nonionic strates were prepared as described (27). The linear oligonucle- detergent)͞5% glycerol͞5 mM NaHSO3), loaded on a 0.8-ml otide templates contained a biotin at both ends, to which a 2-fold MiniQ column in B plus 100 mM NaCl, and eluted by using a 100 molar excess of streptavidin was added. They are V9, 5Ј- to 500 mM NaCl gradient in buffer B. PCNAUbi eluted at Ϸ260 Bio-CCTTTGCGAATTCT25GCGGCTCCCTTCTTCTC- mM NaCl before unmodified