Mammalian base excision repair by DNA polymerases δ and ε

Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Zürich, Zurich, Switzerland
Oncogene (Impact Factor: 8.46). 09/1998; 17(7):835-43. DOI: 10.1038/sj.onc.1202001
Source: PubMed


Two distinct pathways for completion of base excision repair (BER) have been discovered in eukaryotes: the DNA polymerase beta (Pol beta)-dependent short-patch pathway that involves the replacement of a single nucleotide and the long-patch pathway that entails the resynthesis of 2-6 nucleotides and requires PCNA. We have used cell extracts from Pol beta-deleted mouse fibroblasts to separate subfractions containing either Pol delta or Pol epsilon. These fractions were then tested for their ability to perform both short- and long-patch BER in an in vitro repair assay, using a circular DNA template, containing a single abasic site at a defined position. Remarkably, both Pol delta and Pol epsilon were able to replace a single nucleotide at the lesion site, but the repair reaction is delayed compared to single nucleotide replacement by Pol beta. Furthermore, our observations indicated, that either Pol delta and/or Pol epsilon participate in the long-patch BER. PCNA and RF-C, but not RP-A are required for this process. Our data show for the first time that Pol delta and/or Pol epsilon are directly involved in the long-patch BER of abasic sites and might function as back-up system for Pol beta in one-gap filling reactions.

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    • "APEs introduce a nick 5 0 of the lesion (Matsumoto et al. 1994; Klungland & Lindahl 1997). High-fidelity polymerases will synthesize a stretch of several nucleotides while displacing the old strand (Klungland & Lindahl 1997; Stucki et al. 1998). The resulting flap, a stretch of ssDNA, is then removed by flap endonuclease 1 (FEN1; Klungland & Lindahl 1997; Kim et al. 1998). "
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    ABSTRACT: Proliferating cell nuclear antigen (PCNA) encircles DNA as a ring-shaped homotrimer and, by tethering DNA polymerases to their template, PCNA serves as a critical replication factor. In contrast to high-fidelity DNA polymerases, the activation of low-fidelity translesion synthesis (TLS) DNA polymerases seems to require damage-inducible monoubiquitylation (Ub) of PCNA at lysine residue 164 (PCNA-Ub). TLS polymerases can tolerate DNA damage, i.e. they can replicate across DNA lesions. The lack of proofreading activity, however, renders TLS highly mutagenic. The advantage is that B cells use mutagenic TLS to introduce somatic mutations in immunoglobulin (Ig) genes to generate high-affinity antibodies. Given the critical role of PCNA-Ub in activating TLS and the role of TLS in establishing somatic mutations in immunoglobulin genes, we analysed the mutation spectrum of somatically mutated immunoglobulin genes in B cells from PCNAK164R knock-in mice. A 10-fold reduction in A/T mutations is associated with a compensatory increase in G/C mutations-a phenotype similar to Poleta and mismatch repair-deficient B cells. Mismatch recognition, PCNA-Ub and Poleta probably act within one pathway to establish the majority of mutations at template A/T. Equally relevant, the G/C mutator(s) seems largely independent of PCNAK(164) modification.
    Philosophical Transactions of The Royal Society B Biological Sciences 12/2008; 364(1517):621-9. DOI:10.1098/rstb.2008.0223 · 7.06 Impact Factor
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    • "The proliferating cell nuclear antigen (PCNA) reportedly controls the LPR pathway. PCNA is loaded by the replication factor C (RFC) and allows the replicative DNA polymerases δ/ɛ to be clamped in place (16,17). PCNA also stimulates the activity of endonuclease I (FEN-I) to remove flaps (18), and recruits DNA ligase I (Lig I) (19,20). "
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    ABSTRACT: The consequences of PARP-1 disruption or inhibition on DNA single-strand break repair (SSBR) and radio-induced lethality were determined in synchronized, isogenic HeLa cells stably silenced or not for poly(ADP-ribose) polymerase-1 (PARP-1) (PARP-1(KD)) or XRCC1 (XRCC1(KD)). PARP-1 inhibition prevented XRCC1-YFP recruitment at sites of 405 nm laser micro irradiation, slowed SSBR 10-fold and triggered the accumulation of large persistent foci of GFP-PARP-1 and GFP-PCNA at photo damaged sites. These aggregates are presumed to hinder the recruitment of other effectors of the base excision repair (BER) pathway. PARP-1 silencing also prevented XRCC1-YFP recruitment but did not lengthen the lifetime of GFP-PCNA foci. Moreover, PARP-1(KD) and XRCC1(KD) cells in S phase completed SSBR as rapidly as controls, while SSBR was delayed in G1. Taken together, the data demonstrate that a PARP-1- and XRCC1-independent SSBR pathway operates when the short patch repair branch of the BER is deficient. Long patch repair is the likely mechanism, as GFP-PCNA recruitment at photo-damaged sites was normal in PARP-1(KD) cells. PARP-1 silencing elicited hyper-radiosensitivity, while radiosensitization by a PARP inhibitor reportedly occurs only in those cells treated in S phase. PARP-1 inhibition and deletion thus have different outcomes in terms of SSBR and radiosensitivity.
    Nucleic Acids Research 08/2008; 36(13):4454-64. DOI:10.1093/nar/gkn403 · 9.11 Impact Factor
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    • "For more details, see text. carried out by Pol δ or Pol ε, both replicative DNA polymerases (Fortini et al., 1998; Stucki et al., 1998). The resulting " flap " structure is then removed by the endonuclease FEN1 via a singlestranded break (SSB) and, subsequently, the nick is sealed by LigI (Levin et al., 1997). "
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    ABSTRACT: For all living organisms, genome stability is important, but is also under constant threat because various environmental and endogenous damaging agents can modify the structural properties of DNA bases. As a defense, organisms have developed different DNA repair pathways. Base excision repair (BER) is the predominant pathway for coping with a broad range of small lesions resulting from oxidation, alkylation, and deamination, which modify individual bases without large effect on the double helix structure. As, in mammalian cells, this damage is estimated to account daily for 10(4) events per cell, the need for BER pathways is unquestionable. The damage-specific removal is carried out by a considerable group of enzymes, designated as DNA glycosylases. Each DNA glycosylase has its unique specificity and many of them are ubiquitous in microorganisms, mammals, and plants. Here, we review the importance of the BER pathway and we focus on the different roles of DNA glycosylases in various organisms.
    Critical Reviews in Biochemistry and Molecular Biology 07/2008; 43(4):239-76. DOI:10.1080/10409230802309905 · 7.71 Impact Factor
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