Ubiquitination of PCNA and its essential role in eukaryotic translesion synthesis.
ABSTRACT Ubiquitin and ubiquitin-like proteins (Ubls) are now at the center stage of molecular and cell biology because of their diverse functions in many fundamentally important cellular processes. Besides the celebrated role of ubiquitin in the 26S proteasome-mediated protein degradation pathway, the non-proteolytic functions of ubiquitin are being uncovered at a fast pace. The prominent examples include membrane trafficking, innate immunity, kinase signaling, chromatin dynamics and DNA damage response. Researchers in the area of DNA damage response have witnessed rapid progress within the past decade, largely stimulated by the seminal findings that ubiquitination and SUMOylation of a key DNA replication/repair protein, proliferating cell nuclear antigen (PCNA), controls precisely how eukaryotic cells respond to different types of DNA damage, and how cellular DNA damage repair or tolerance pathways are selected to cope with damage in the DNA genome. Here, we will review the recent findings on translesion synthesis (TLS) and its regulation by PCNA ubiquitination in eukaryotes. We will discuss two prevalent models, i.e., the postreplicative gap-filling and the polymerase switch, which have been invoked to account for eukaryotic cells' ability to overcome DNA damage associated replication blockade through TLS. Results from both in vitro reconstitution and from genetic systems will be discussed. We will also summarize the recent findings revealing the crosstalk between two major human DNA damage response pathways (the TLS and the Fanconi anemia pathways), and the ATR and ATM-independent regulation of PCNA ubiquitination. Lastly, new methods of preparing ubiquitinated PCNA will be reviewed. The availability of milligram levels of ubiquitinated PCNA will help our understanding of the molecular details in eukaryotic TLS.
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ABSTRACT: Trans-lesion DNA polymerases (TLSPs) enable bypass of DNA lesions during replication and are also induced under stress conditions. Being only weakly dependent on their template during replication, TLSPs introduce mutations into DNA. The low processivity of these enzymes ensures that they fall off their template after a few bases are synthesized and are then replaced by the more accurate replicative polymerase. We find that the three TLSPs of budding yeast Saccharomyces cerevisiae Rev1, PolZeta (Rev3 and Rev7), and Rad30 are induced during meiosis at a time when DNA double-strand breaks (DSBs) are formed and homologous chromosomes recombine. Strains deleted for one or any combination of the three TLSPs undergo normal meiosis. However, in the triple-deletion mutant, there is a reduction in both allelic and ectopic recombination. We suggest that trans-lesion polymerases are involved in the processing of meiotic double-strand breaks that lead to mutations. In support of this notion, we report significant yeast two-hybrid (Y2H) associations in meiosis-arrested cells between the TLSPs and DSB proteins Rev1-Spo11, Rev1-Mei4, and Rev7-Rec114, as well as between Rev1 and Rad30. We suggest that the involvement of TLSPs in processing of meiotic DSBs could be responsible for the considerably higher frequency of mutations reported during meiosis compared with that found in mitotically dividing cells, and therefore may contribute to faster evolutionary divergence than previously assumed.G3-Genes Genomes Genetics 03/2013; 3(4). DOI:10.1534/g3.113.005603 · 2.51 Impact Factor
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ABSTRACT: The p12 subunit of polymerase delta (Pol δ) is degraded in response to DNA damage induced by UV, alkylating agents, oxidative, and replication stresses. This leads to the conversion of the Pol δ4 holoenzyme to the heterotrimer, Pol δ3. We review studies that establish that Pol δ3 formation is an event that could have a major impact on cellular processes in genomic surveillance, DNA replication, and DNA repair. p12 degradation is dependent on the apical ataxia telangiectasia and Rad3 related (ATR) kinase and is mediated by the ubiquitin-proteasome system. Pol δ3 exhibits properties of an "antimutator" polymerase, suggesting that it could contribute to an increased surveillance against mutagenesis, for example, when Pol δ carries out bypass synthesis past small base lesions that engage in spurious base pairing. Chromatin immunoprecipitation analysis and examination of the spatiotemporal recruitment of Pol δ to sites of DNA damage show that Pol δ3 is the primary form of Pol δ associated with cyclobutane pyrimidine dimer lesions and therefore should be considered as the operative form of Pol δ engaged in DNA repair. We propose a model for the switching of Pol δ with translesion polymerases, incorporating the salient features of the recently determined structure of monoubiquitinated proliferating cell nuclear antigen and emphasizing the role of Pol δ3. Because of the critical role of Pol δ activity in DNA replication and repair, the formation of Pol δ3 in response to DNA damage opens the prospect that pleiotropic effects may ensue. This opens the horizons for future exploration of how this novel response to DNA damage contributes to genomic stability. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.Environmental and Molecular Mutagenesis 12/2012; 53(9). DOI:10.1002/em.21743 · 2.55 Impact Factor
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