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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    • "In the SP-BER, after the dNMP insertion, the deoxyribose-phosphate (dRP) is removed by the dRP-lyase activity of Polí µí»½[75]and the repair is completed by ligation by the Lig3/XRCC1 complex. In LP- BER, the dRP is displaced and Polí µí»½/Polí µí»¿ polymerize tracts of DNA longer than one base[71,76]. The strand displacement produces a flapped substrate that is refractory to ligation; this structure is recognized and excised by FEN1[77,78], followed by ligation by DNA ligase 1 (Lig1)[79]. "
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    ABSTRACT: There is a growing body of evidence indicating that the mechanisms that control genome stability are of key importance in the development and function of the nervous system. The major threat for neurons is oxidative DNA damage, which is repaired by the base excision repair (BER) pathway. Functional mutations of enzymes that are involved in the processing of single-strand breaks (SSB) that are generated during BER have been causally associated with syndromes that present important neurological alterations and cognitive decline. In this review, the plasticity of BER during neurogenesis and the importance of an efficient BER for correct brain function will be specifically addressed paying particular attention to the brain region and neuron-selectivity in SSB repair-associated neurological syndromes and age-related neurodegenerative diseases.
    Full-text · Article · Feb 2016 · Neural Plasticity
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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.
    Full-text · Article · Dec 2008 · Philosophical Transactions of The Royal Society B Biological Sciences
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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-1KD) or XRCC1 (XRCC1KD). 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-1KD and XRCC1KD 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-1KD 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.
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