Termination of DNA synthesis in vitro at apurinic sites but not at ethyl adducts on the template

Lockhart, Michael L.; Deutsch, J.F.; Yamaura, Izumi; Cavalieri, Liebe F.; Rosenberg, Barbara Hatch

doi:10.1016/0009-2797(82)90144-2

Cited by 20 publications

(8 citation statements)

References 16 publications

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“…Interestingly, the intermediate AP sites and SSB generated in this pathway are more cytotoxic in many cases than the modified base lesions themselves (Chaudhry and Weinfeld, 1997;Lockhart et al, 1982;Loeb and Preston, 1986;Sagher and Strauss, 1983;Schaaper et al, 1983;Whitehouse et al, 2001). MNU was used in the present studies to generate alkylation damage.…”

Section: Discussionmentioning

confidence: 99%

Altering DNA base excision repair: Use of nuclear and mitochondrial‐targeted N‐methylpurine DNA glycosylase to sensitize astroglia to chemotherapeutic agents

Harrison

Rinne

Kelley

et al. 2007

Glia

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Primary astrocyte cultures were used to investigate the modulation of DNA repair as a tool for sensitizing astrocytes to genotoxic agents. Base excision repair (BER) is the principal mechanism by which mammalian cells repair alkylation damage to DNA and involves the processing of relatively nontoxic DNA adducts through a series of cytotoxic intermediates during the course of restoring normal DNA integrity. An adenoviral expression system was employed to target high levels of the BER pathway initiator, N-methylpurine glycosylase (MPG), to either the mitochondria or nucleus of primary astrocytes to test the hypothesis that an alteration in BER results in increased alkylation sensitivity. Increasing MPG activity significantly increased BER kinetics in both the mitochondria and nuclei. Although modulating MPG activity in mitochondria appeared to have little effect on alkylation sensitivity, increased nuclear MPG activity resulted in cell death in astrocyte cultures treated with methylnitrosourea (MNU). Caspase-3 cleavage was not detected, thus indicating that these alkylation sensitive astrocytes do not undergo a typical programmed cell death in response to MNU. Astrocytes were found to express relatively high levels of antiapoptotic Bcl-2 and Bcl-XL and very low levels of proapoptotic Bad and Bid suggesting that the mitochondrial pathway of apoptosis may be blocked making astrocytes less vulnerable to proapoptotic stimuli compared with other cell types. Consequently, this unique characteristic of astrocytes may be responsible, in part, for resistance of astrocytomas to chemotherapeutic agents.

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Section: Discussionmentioning

confidence: 99%

Altering DNA base excision repair: Use of nuclear and mitochondrial‐targeted N‐methylpurine DNA glycosylase to sensitize astroglia to chemotherapeutic agents

Harrison

Rinne

Kelley

et al. 2007

Glia

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“…This poses a threat since abasic sites act as a block to numerous essential cellular processes, including transcription and replication. When replicative polymerases encounter abasic sites, they pause at the lesion, ultimately stalling the replication fork at a 3′ dsDNA-ssDNA primer-template junction (PTJ) [12][13][14][15][16] . However, how abasic sites at stalled replication forks are targeted to distinct bypass/repair pathways remains unknown.…”

Section: Introductionmentioning

confidence: 99%

Mechanistic Insight into AP-Endonuclease 1 Cleavage of Abasic Sites at Stalled Replication Forks

Hoitsma

Norris

Khoang

et al. 2022

Preprint

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Many types of DNA damage stall replication fork progression, including abasic sites. AP-Endonuclease 1 (APE1) has been shown to cleave abasic sites in ssDNA substrates. Importantly, APE1 cleavage of ssDNA at a replication fork has significant biological implications by generating double strand breaks that could collapse the replication fork. Despite this, the molecular basis and efficiency of APE1 processing abasic sites at a replication fork remains elusive. Here, we investigate APE1 cleavage of several abasic substrates that mimic potential APE1 interactions at replication forks. We determine that APE1 has robust activity on these substrates, similar to dsDNA, and report rapid rates for cleavage and product release. X-ray crystal structures visualize the APE1 active site, highlighting that a similar mechanism is used to process ssDNA substrates as canonical APE1 activity on dsDNA. However, mutational analysis reveals R177 to be uniquely critical for the APE1 ssDNA cleavage mechanism. Additionally, we investigate the interplay between APE1 and Replication Protein A (RPA), the major ssDNA-binding protein at replication forks, revealing that APE1 can cleave an abasic site while RPA is still bound to the DNA substrate. Together, this work provides molecular level insights into abasic ssDNA processing by APE1, including the presence of RPA.

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“…BER involves the recognition and removal of damaged bases by a DNA glycosylase, followed by incision of the resulting abasic (AP) site by AP endonuclease (Ape 1), DNA synthesis by polymerase and strand ligation by DNA ligase (4). This pathway, therefore, involves the processing of modest structural DNA damage through toxic baseless and strand-interrupted species (5–7), which may transiently replace less toxic or non-toxic DNA lesions in the course of restoring the DNA sequence.…”

Section: Introductionmentioning

confidence: 99%

N-methylpurine DNA glycosylase overexpression increases alkylation sensitivity by rapidly removing non-toxic 7-methylguanine adducts

Rinne

Pachkowski³

et al. 2005

Nucleic Acids Research

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Previous studies indicate that overexpression of N-methylpurine DNA glycosylase (MPG) dramatically sensitizes cells to alkylating agent-induced cytotoxicity. We recently demonstrated that this sensitivity is preceded by an increased production of AP sites and strand breaks, confirming that overexpression of MPG disrupts normal base excision repair and causes cell death through overproduction of toxic repair intermediates. Here we establish through site-directed mutagenesis that MPG-induced sensitivity to alkylation is dependent on enzyme glycosylase activity. However, in contrast to the sensitivity seen to heterogeneous alkylating agents, MPG overexpression generates no cellular sensitivity to MeOSO2(CH2)2-lexitropsin, an alkylator which exclusively induces 3-meA lesions. Indeed, MPG overexpression has been shown to increase the toxicity of alkylating agents that produce 7-meG adducts, and here we demonstrate that MPG-overexpressing cells have dramatically increased removal of 7-meG from their DNA. These data suggest that the mechanism of MPG-induced cytotoxicity involves the conversion of non-toxic 7-meG lesions into highly toxic repair intermediates. This study establishes a mechanism by which a benign DNA modification can be made toxic through the overexpression of an otherwise well-tolerated gene product, and the application of this principle could lead to improved chemotherapeutic strategies that reduce the peripheral toxicity of alkylating agents.

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Termination of DNA synthesis in vitro at apurinic sites but not at ethyl adducts on the template

Cited by 20 publications

References 16 publications

Altering DNA base excision repair: Use of nuclear and mitochondrial‐targeted N‐methylpurine DNA glycosylase to sensitize astroglia to chemotherapeutic agents

Altering DNA base excision repair: Use of nuclear and mitochondrial‐targeted N‐methylpurine DNA glycosylase to sensitize astroglia to chemotherapeutic agents

Mechanistic Insight into AP-Endonuclease 1 Cleavage of Abasic Sites at Stalled Replication Forks

N-methylpurine DNA glycosylase overexpression increases alkylation sensitivity by rapidly removing non-toxic 7-methylguanine adducts

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