Efficiency of Repair of an Abasic Site within DNA Clustered Damage Sites by Mammalian Cell Nuclear Extracts

Lomax, Martine E.; Cunniffe, Siobhan; O’Neill, Peter

doi:10.1021/bi049560r

Cited by 86 publications

(102 citation statements)

References 34 publications

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“…These damaged templates undergo rapid BER incisional activity that subsequently result in increased DSBs [49]. Others have also shown that clustered BER sites on double-stranded DNA can give rise to DSBs by incisional repair activity [59]. Another recent study has demonstrated that MNNG induces replicationindependent DSBs in a dose and MMR-dependent manner, and does not require DNA replication.…”

Section: Discussionmentioning

confidence: 99%

Rapid induction of chromatin-associated DNA mismatch repair proteins after MNNG treatment

Schroering

Williams

2008

DNA Repair

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Treatment with low concentrations of monofunctional alkylating agents induces a G 2 arrest only after the second round of DNA synthesis in mammalian cells and requires a proficient mismatch repair (MMR) pathway. Here we have investigated rapid alkylation-induced recruitment of DNA repair proteins to chromosomal DNA within synchronized populations of MMR proficient cells (HeLa MR) after MNNG treatment. Within the first hour, the concentrations of MutSα and PCNA increase well beyond their constitutive chromosomally bound levels and MutLα is newly recruited to the chromatin-bound MutSα. Remarkably, immunoprecipitation experiments demonstrate rapid association of these proteins on the alkylation-damaged chromatin, even when DNA replication is completely blocked. The extent of association of PCNA and MMR proteins on the chromatin is dependent upon the concentration of MNNG and on the specific type of replication block. A subpopulation of the MutSα-associated PCNA also becomes monoubiquitinated, a known requirement for PCNA to interact with translesion synthesis (TLS) polymerases. In addition, chromatin-bound SMC1 and NBS1 proteins, associated with DNA double-strand-breaks (DSBs), become phosphorylated within one to two hours of exposure to MNNG. However, these activated proteins are not colocalized on the chromatin with MutSα in response to MNNG exposure. PCNA, MutSα/MutLα and activated SMC1/NBS1 remain chromatin-bound for at least 6-8 hours after alkylation damage. Thus, cells that are exposed to low levels of alkylation treatment undergo rapid recruitment to and/or activation of key proteins already on the chromatin without the requirement for DNA replication, apparently via different DNA-damage signaling pathways.

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Section: Discussionmentioning

confidence: 99%

Rapid induction of chromatin-associated DNA mismatch repair proteins after MNNG treatment

Schroering

Williams

2008

DNA Repair

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“…The time course performed on the tandem lesions (3,4) was carried out in the same way as 1 except the 2× enzyme concentration was 200 nM, the 2× DNA solution was 2 nM, and aliquots were removed at 0, 10, 30, 60, 180, 300, 900, 1800, and 3600 s. The time course experiments for the clustered lesion containing duplexes (5,6) were run with a 2× DNA concentration of 2 nM and 2× enzyme concentration of 200 nM. Aliquots were removed at 0, 60, 180, 300, 900, 1800, and 3600 s.…”

Section: Time Course For Nth Excision Of Thymine Glycolmentioning

confidence: 99%

“…Aliquots (1.5 μL) were removed at 0, 10, 30, 60, 180, 300, 900, and 1800 s. Experiments using the clustered lesion containing duplexes (5,6) were carried out in the same way as 2 except aliquots (1.5 μL) were removed at 0, 10, 30, 60, 180, 300, 900, and 1800 s. For experiments with the tandem lesion (3), 2× DNA concentrations used were 50, 100, 150, 250, 500, and 1000 nM, and the reaction time was 3 min. For experiments with the clustered lesion containing duplexes (5,6) the 2× DNA concentrations used were 20, 50, 100, 150, 250, and 400 nM, and the reaction time was 1 min.…”

Section: Time Course For Nfo Incision Of 2-deoxyribonolactone (L) or mentioning

confidence: 99%

DNA Tandem Lesion Repair by Strand Displacement Synthesis and Nucleotide Excision Repair

et al. 2008

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DNA tandem lesions are comprised of two contiguously damaged nucleotides. This subset of clustered lesions is produced by a variety of oxidizing agents, including ionizing radiation. Clustered lesions can inhibit base excision repair (BER). We report the effects of tandem lesions composed of a thymine glycol and a 5′-adjacent 2-deoxyribonolactone (LTg) or tetrahydrofuran abasic site (FTg). Some BER enzymes that act on the respective isolated lesions do not accept the tandem lesion as a substrate. For instance, endonuclease III (Nth) does not excise thymine glycol (Tg) when it is part of either tandem lesion. Similarly, endonuclease IV (Nfo) does not incise L or F when they are in tandem with Tg. Long-patch BER overcomes inhibition by the tandem lesion. DNA polymerase β (Pol β) carries out strand displacement synthesis, following APE1 incision of the abasic site. Pol β activity is enhanced by flap endonuclease (FEN1), which cleaves the resulting flap. The tandem lesion is also incised by the bacterial nucleotide excision repair system UvrABC with almost the same efficiency as an isolated Tg. These data reveal two solutions that DNA repair systems can use to counteract the formation of tandem lesions.Isolated damaged nucleotides are believed to be the most common lesions resulting from DNA oxidation. As a result of efforts by many researchers, a great deal has been learned about how DNA lesions are repaired by base excision repair (BER) 1 and nucleotide excision repair (NER) (1-3). Recently, clusters of DNA lesions have been identified as significant components of radiation-induced damage. Clustered lesions consist of two or more damaged nucleotides within ~1.5 turns of duplex DNA and are attributed to multiple ionization events by a single radiation track. In principle, clustered lesions can contain modified nucleotides on a single strand, but biochemical studies have focused on bistranded lesions, which are possible progenitors to double strand breaks (1-11). Numerous studies have revealed the effects of clustered lesions on BER. The effects are dependent upon the nature of the lesions, as well as their proximity to one another in duplex DNA. Clustered lesions consisting of two contiguously damaged nucleotides are referred to as tandem lesions (12,13). Unlike other examples of clustered damage, tandem lesions can result from a single DNA damaging event, in which a † We are grateful for support of this research by the National Institute of General Medical Science (Grant GM-063028). This research was supported in part by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.* To whom correspondence should be addressed. . E-mail: mgreenberg@jhu.edu. ‡ These researchers contributed equally to the research described in this paper. § Johns Hopkins University. || Laboratory of Molecular Genetics, NIEHS, NIH.1 Abbreviations: AP, abasic site; L, 2-deoxyribonolactone; F, tetrahydrofuran abasic site; Tg, thymine glycol; BER, base excision repair; LP-BER, long-patch base exc...

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“…Whereas both ATM activation and ␥H2AX are correlated with the presence of double-strand breaks (DSBs) in the genome (Abraham, 2001;Pilch et al, 2003), these biochemical events indicate that DSBs occur in response to high-dose MNNG exposure. DSBs generated by alkylators are caused by the simultaneous processing of clustered AP sites on opposite DNA strands (Lomax et al, 2004), and their abundance would be expected to be proportional to initial lesion load. However, given the ATM-independent nature of high-dose MNNG G 2 checkpoint activation, it is unlikely that MNNG-induced DSBs are the dominant lesion that triggers activation of this checkpoint.…”

Section: Mnng-induced Checkpoint Signaling: High-versusmentioning

confidence: 99%

N-Methyl-N′-nitro-N-nitrosoguanidine Activates Cell-Cycle Arrest through Distinct Mechanisms Activated in a Dose-Dependent Manner

Beardsley¹,

Kim²,

Brown³

2005

Mol Pharmacol

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S N 1-alkylating agents, such as the mutagenic and cytotoxic drug N-methyl- NЈ-nitro-N-nitrosoguanidine (MNNG), robustly activate the DNA damage-responsive G 2 checkpoint. Establishment of this checkpoint is dependent on a functional mismatch repair (MMR) system; however, exposure to high doses of MNNG overrides the requirement for MMR to trigger G 2 arrest. In addition, unlike moderate-dose exposure, in which the G 2 checkpoint is attenuated in ataxia-telangiectasia, mutated (ATM)-deficient cells, high-dose MNNG treatment activates G 2 arrest through an ATM-independent mechanism. We document that this arrest is sensitive to the pharmacological agents caffeine and 7-hydroxystaurosporine (UCN-01) that inhibit the checkpoint kinases ATM/ATM and Rad-3-related (ATR) and Chk1/Chk2, respectively. Furthermore, these agents block inactivation of the cell-cycle regulatory molecules Cdc25C and Cdc2, establishing the downstream mechanism through which high-dose MNNG establishes G 2 arrest. Activation of both Chk2 and Chk1 was independent of ATM and MMR in response to high-dose MNNG, unlike the response to moderate doses of this drug. Chk2 was found to be dispensable for cell-cycle arrest in response to high-dose MNNG treatment; however, ATR deficiency and decreased Chk1 expression forced by RNA interference resulted in diminished checkpoint response. These results indicate that MNNG activates the G 2 checkpoint through different mechanisms activated in a dosedependent fashion.

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Efficiency of Repair of an Abasic Site within DNA Clustered Damage Sites by Mammalian Cell Nuclear Extracts

Cited by 86 publications

References 34 publications

Rapid induction of chromatin-associated DNA mismatch repair proteins after MNNG treatment

Rapid induction of chromatin-associated DNA mismatch repair proteins after MNNG treatment

DNA Tandem Lesion Repair by Strand Displacement Synthesis and Nucleotide Excision Repair

N-Methyl-N′-nitro-N-nitrosoguanidine Activates Cell-Cycle Arrest through Distinct Mechanisms Activated in a Dose-Dependent Manner

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