Dynamics and diversions in base excision DNA repair of oxidized abasic lesions

Demple, Bruce; DeMott, Michael S.

doi:10.1038/sj.onc.1206178

Cited by 94 publications

(64 citation statements)

References 110 publications

(97 reference statements)

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“…This sensitivity profile of the ED-expressing cells is consistent with APE1 exhibiting a robust AP endonuclease activity in vitro (even for oxidized AP sites ref. 35), yet a comparably weak 3 ¶-phosphodiesterase function (36), and with the polynucleotide kinase/phosphatase protein operating as the main human DNA 3 ¶-phosphatase (37). The data also suggest that AP sites are effective cytotoxic lesions, a conclusion that is consistent with the observation that sufficient APE1 depletion in mammalian cells leads to an accumulation of AP sites and an accompanying cell inviability (15).…”

Section: Discussionsupporting

confidence: 75%

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

McNeill

Wilson

2007

Molecular Cancer Research

109

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Apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is the primary enzyme in mammals for the repair of abasic sites in DNA, as well as a variety of 3 ¶ damages that arise upon oxidation or as products of enzymatic processing. If left unrepaired, APE1 substrates can promote mutagenic and cytotoxic outcomes. We describe herein a dominant-negative form of APE1 that lacks detectable nuclease activity and binds substrate DNA with a 13-fold higher affinity than the wild-type protein. This mutant form of APE1, termed ED, possesses two amino acid substitutions at active site residues Glu 96 (changed to Gln) and Asp 210 (changed to Asn). In vitro biochemical assays reveal that ED impedes wild-type APE1 AP site incision function, presumably by binding AP-DNA and blocking normal lesion processing. Moreover, tetracycline-regulated (tet-on) expression of ED in Chinese hamster ovary cells enhances the cytotoxic effects of the laboratory DNA-damaging agents, methyl methanesulfonate (MMS; 5.4-fold) and hydrogen peroxide (1.5-fold). This MMS-induced, ED-dependent cell killing coincides with a hyperaccumulation of AP sites, implying that excessive DNA damage is the cause of cell death. Because an objective of the study was to identify a protein reagent that could be used in targeted gene therapy protocols, the effects of ED on cellular sensitivity to a number of chemotherapeutic compounds was tested. We show herein that ED expression sensitizes Chinese hamster ovary cells to the killing effects of the alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (also known as carmustine) and the chain terminating nucleoside analogue dideoxycytidine (also known as zalcitabine), but not to the radiomimetic bleomycin, the nucleoside analogue B-D-arabinofuranosylcytosine (also known as cytarabine), the topoisomerase inhibitors camptothecin and etoposide, or the cross-linking agents mitomycin C and cisplatin. Transient expression of ED in the human cancer cell line NCI-H1299 enhanced cellular sensitivity to MMS, 1,3-bis(2-chloroethyl)-1-nitrosourea, and dideoxycytidine, demonstrating the potential usefulness of this strategy in the treatment of human tumors.

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Section: Discussionsupporting

confidence: 75%

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

McNeill

Wilson

2007

Molecular Cancer Research

109

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“…AP sites could be further oxidized to various products with oxidation and fragmentation of deoxyribose moiety and strand cleavage [87]. The major products include 2′-deoxyribonolactone, 2-deoxypentos-4-ulose and 3′ phosphoglycolate [14]. Unmodified AP sites are also generated by monofunctional DNA glycosylases, as already mentioned.…”

Section: Two Basic Modes Of Ber: Sn-vs Lp-bermentioning

confidence: 99%

“…The 3′ blocking groups include 3′ phosphate, 3′ phosphoglycolaldehyde, or 3′ phosphoglycolate [13]. The 5′ terminus normally contains phosphate but after ROS reaction the nonligatable ends include 5′ OH and 5′ phosphodeoxyribose derivatives such as 2-deoxyribonolactone [14].…”

Section: Base Excision Repair (Ber)mentioning

confidence: 99%

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

2008

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Base excision repair (BER) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ROS). BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease (APE) to generate 3′ OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA ligase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APE1, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3′ phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and ligases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organelle targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.

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“…Thus nfo and xth mutants also are sensitive to peroxides (89,90), and the double mutant is marginally viable in aerobic media. Interestingly, while eukaryotes do not contain genetic orthologues of these enzymes, they have functional analogues, and mutants that lack them are also hypersensitive to oxidants (91).…”

Section: Dna Repairmentioning

confidence: 99%

Cellular Defenses against Superoxide and Hydrogen Peroxide

Imlay

2008

Annu. Rev. Biochem.

1,335

1,310

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Life evolved in an anaerobic world; therefore, fundamental enzymatic mechanisms and biochemical pathways were refined and integrated into metabolism in the absence of any selective pressure to avoid reactivity with oxygen. After photosystem 2 appeared, environmental oxygen levels rose very slowly. During this time microorganisms acquired oxygen tolerance by jettisoning enzymes that use glycyl radicals and low-potential iron-sulfur clusters, which can be directly poisoned by oxygen. They also developed mechanisms to defend themselves against O 2 − and hydrogen peroxide, partially reduced oxygen species that are generated as inadvertent by-products of aerobic metabolism. These species are more chemically reactive than is molecular oxygen itself. Contemporary organisms have inherited both the vulnerabilities and the defenses of these ancestral microbes. Current research seeks to identify these, and bacteria comprise an exceptionally accessible experimental system that has provided the many of the answers. This manuscript reviews recent developments and identifies remaining puzzles.

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Dynamics and diversions in base excision DNA repair of oxidized abasic lesions

Cited by 94 publications

References 110 publications

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

Cellular Defenses against Superoxide and Hydrogen Peroxide

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