Role for DNA polymerase beta in response to ionizing radiation

Vermeulen, Christie; Verwijs-Janssen, Manon; Cramers, Patricia; Begg, Adrian C.; Vens, Conchita

doi:10.1016/j.dnarep.2006.09.011

Cited by 18 publications

(22 citation statements)

References 46 publications

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“…Alternatively, the long-patch repair DNA polymerases Pol ␦ and/or Pol ε might substitute for Pol ␤. Consistent with this idea, while early-passage and proliferating populations of Pol ␤ Ϫ/Ϫ murine embryonic fibroblasts exhibit normal sensitivity and DNA strand break repair rates following oxidative stress, increased sensitivity and decreased repair rates are observed in late-passage or G 1 -enriched Pol ␤ Ϫ/Ϫ murine embryonic fibroblasts, conditions under which longpatch repair machinery may be downregulated (21,52,53). It remains to be determined whether a similar cell cycle stagespecific requirement exists for the interaction of Pol ␤ with XRCC1.…”

Section: Discussionsupporting

confidence: 51%

DNA 3′-Phosphatase Activity Is Critical for Rapid Global Rates of Single-Strand Break Repair following Oxidative Stress

Breslin

Caldecott

2009

Molecular and Cellular Biology

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Oxidative stress is a major source of chromosome single-strand breaks (SSBs), and the repair of these lesions is retarded in neurodegenerative disease. The rate of the repair of oxidative SSBs is accelerated by XRCC1, a scaffold protein that is essential for embryonic viability and that interacts with multiple DNA repair proteins. However, the relative importance of the interactions mediated by XRCC1 during oxidative stress in vivo is unknown. We show that mutations that disrupt the XRCC1 interaction with DNA polymerase ␤ or DNA ligase III fail to slow SSB repair in proliferating CHO cells following oxidative stress. In contrast, mutation of the domain that interacts with polynucleotide kinase/phosphatase (PNK) and Aprataxin retards repair, and truncated XRCC1 encoding this domain fully supports this process. Importantly, the impact of mutating the protein domain in XRCC1 that binds these end-processing factors is circumvented by the overexpression of wild-type PNK but not by the overexpression of PNK harboring a mutated DNA 3-phosphatase domain. These data suggest that DNA 3-phosphatase activity is critical for rapid rates of chromosomal SSB repair following oxidative stress, and that the XRCC1-PNK interaction ensures that this activity is not rate limiting in vivo.Oxidative stress can have a major influence on genome integrity and cell survival and is an etiological factor in a number of neurological human diseases. Of these, several are associated with defects in the repair of DNA damage, including xeroderma pigmentosum (XP), ataxia telangiecatsia (A-T), ataxia oculomotor apraxia 1 (AOA1), and spinocerebellar ataxia with axonal neuropathy 1 (SCAN1) (1,28,34,37). The neuropathology evident in XP most likely reflects an inability to repair one or more single-strand oxidative adducts by nucleotide excision repair. In contrast, A-T is associated with cellular defects in the repair of DNA double-strand breaks (DSBs) and AOA1 and SCAN1 with defects in the repair of DNA single-strand breaks (SSBs). SSBs are the commonest DNA lesions arising in cells, and if they are not rapidly repaired they can inhibit transcription and/or generate replication-associated DSBs (3,26,59,60). The repair of oxidative SSBs involves DNA damage detection by PARP-1 followed by recruitment of the enzymes required for subsequent steps of the repair process, which include DNA end processing, DNA gap filling, and DNA ligation (9, 19). Many of the enzymes implicated in these steps interact physically with XRCC1, including DNA polynucleotide kinase (PNK) (54), Aprataxin (APTX) (13,14,18,31,44), DNA polymerase ␤ (Pol ␤) (10, 27), and DNA ligase III␣ (Lig3␣) (11,12). This has prompted the hypothesis that XRCC1 is a scaffold protein that recruits, stabilizes, and/or stimulates SSB repair (SSBR) enzymes at chromosomal SSBs, thereby accelerating the overall process (8, 9). While in vitro analyses generally are consistent with this idea, including the observation that XRCC1 mutation (50, 58), deletion (49), or depletion (6) retards the rate of chromos...

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Section: Discussionsupporting

confidence: 51%

DNA 3′-Phosphatase Activity Is Critical for Rapid Global Rates of Single-Strand Break Repair following Oxidative Stress

Breslin

Caldecott

2009

Molecular and Cellular Biology

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“…AP or dRp lyases) or by high pH [8-10]. Methoxyamine hydrochloride has been used ex vivo as a BER inhibitor [11-13] and in vitro as a stabilizer of AP sites for the detection of damaged bases (e.g. N7 -methylpurines) [14, 15] or AP sites [16, 17].…”

Section: Introductionmentioning

confidence: 99%

“…N7 -methylpurines) [14, 15] or AP sites [16, 17]. However, the acidic methoxyamine hydrochloride must be precisely neutralized [13, 18-20] before the usage. Furthermore, the neutralization of methoxyamine hydrochloride may lead to high salt contamination.…”

Section: Introductionmentioning

confidence: 99%

Accumulation of true single strand breaks and AP sites in base excision repair deficient cells

Luke

Chastain

Pachkowski

et al. 2010

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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Single strand breaks (SSBs) are one of the most frequent DNA lesions caused by endogenous and exogenous agents. The most utilized alkaline-based assays for SSB detection frequently give false positive results due to the presence of alkali-labile sites that are converted to SSBs. Methoxyamine, an acidic O-hydroxylamine, has been utilized to measure DNA damage in cells. However, the neutralization of methoxyamine is required prior to usage. Here we developed a convenient, specific SSB assay using alkaline gel electrophoresis (AGE) coupled with a neutral O-hydroxylamine, O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (OTX). OTX stabilizes abasic sites (AP sites) to prevent their alkaline incision while still allowing for strong alkaline DNA denaturation. DNA from DT40 and isogenic polymerase β null cells exposed to methyl methanesulfonate were applied to the OTX-coupled AGE (OTX-AGE) assay. Time-dependent increases in SSBs were detected in each cell line with more extensive SSB formation in the null cells. These findings were supported by an assay that indirectly detects SSBs through measuring NAD(P)H depletion. An ARP-slot blot assay demonstrated a significant time-dependent increase in AP sites in both cell lines by 1 mM MMS compared to control. Furthermore, the Pol β-null cells displayed greater AP site formation than the parental DT40 cells. OTX use represents a facile approach for assessing SSB formation, whose benefits can also be applied to other established SSB assays.

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“…Alternatively, there might be robust mechanisms for repair of IR-induced DNA damage that, in the absence of pol β, can efficiently substitute for pol β-dependent pathways. A recent study has described IR hypersensitivity in non-dividing pol β null cells suggesting that pol β-dependent repair is masked by alternate replication associated repair pathways in cycling cells [89].…”

Section: Characteristics Of Xrcc1-and Pol β-Deficient Cells: Hypersenmentioning

confidence: 99%

XRCC1 and DNA polymerase β in cellular protection against cytotoxic DNA single-strand breaks

et al. 2008

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Single-strand breaks (SSBs) can occur in cells either directly, or indirectly following initiation of base excision repair (BER). SSBs generally have blocked termini lacking the conventional 5′-phosphate and 3′-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities.

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Role for DNA polymerase beta in response to ionizing radiation

Cited by 18 publications

References 46 publications

DNA 3′-Phosphatase Activity Is Critical for Rapid Global Rates of Single-Strand Break Repair following Oxidative Stress

DNA 3′-Phosphatase Activity Is Critical for Rapid Global Rates of Single-Strand Break Repair following Oxidative Stress

Accumulation of true single strand breaks and AP sites in base excision repair deficient cells

XRCC1 and DNA polymerase β in cellular protection against cytotoxic DNA single-strand breaks

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