In Vitro Replication and Repair of DNA Containing a C2‘-Oxidized Abasic Site

Greenberg, Marc M.; Weledji, Yvonne N.; Kroeger, Kelly M.; Kim, Jaeseung

doi:10.1021/bi048360c

Cited by 25 publications

(28 citation statements)

References 26 publications

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“…The K m for this process is comparable to those describing AP and L incision by Ape1, but the k cat is about 30-fold slower (35). In addition, Ape1 incises C2-AP approximately 10-fold less efficiently than does Xth (8). Pol β efficiently extends the 3′-terminus of the fragment at the incision site by 2 nucleotides, or one nucleotide past the lesion, and is assisted by FEN1.…”

Section: Discussionmentioning

confidence: 98%

Excision of a Lyase-Resistant Oxidized Abasic Lesion from DNA

Wong

Sczepanski

Greenberg

2010

Chem. Res. Toxicol.

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The C2′-oxidized abasic lesion (C2-AP) is produced in DNA that is subjected to oxidative stress. The lesion disrupts replication and gives rise to mutations that are dependent upon the identity of the upstream nucleotide. Ape1 incises C2-AP, but the 5′-phosphorylated fragment is not a substrate for the lyase activity of DNA polymerase β (Pol β). Excision of the lesion is achieved by strand displacement synthesis in the presence of flap endonuclease (FEN1) during which C2-AP and the 3′-adjacent nucleotide are replaced. The oxidized abasic lesion is also a substrate for the bacterial UvrABC nucleotide excision repair system. These data suggest that the redundant nature of DNA repair systems provide a means for removing a lesion that resists excision by short patch base excision repair.

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Section: Discussionmentioning

confidence: 98%

Excision of a Lyase-Resistant Oxidized Abasic Lesion from DNA

Wong

Sczepanski

Greenberg

2010

Chem. Res. Toxicol.

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“…Deoxyribose structures, including chemically reduced abasic sites, abasic site analogues ( e.g. , tetrahydrofuran), and sugar lesions arising from either C-1′ or C-2′ oxidation of deoxyribose that have been initially cleaved on the 5′ side by APE1 are refractory to excision by POLβ (60, 61). Since the chemical structures of these 5′-end lesions prevent β-elimination by POLβ, they will be substrates for FEN1-dependent repair by LP-BER (62).…”

Section: Discussionmentioning

confidence: 99%

FEN1 Functions in Long Patch Base Excision Repair Under Conditions of Oxidative Stress in Vertebrate Cells

Asagoshi

Tano

Chastain

et al. 2010

Molecular Cancer Research

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From in vitro studies, flap endonuclease 1 (FEN1) has been proposed to play a role in the long patch (LP) base excision repair (BER) subpathway. Yet the role of FEN1 in BER in the context of the living vertebrate cell has not been thoroughly explored. In the present study, we cloned a DT40 chicken cell line with a deletion in the FEN1 gene and found that these FEN1-deficient cells exhibited hypersensitivity to H 2 O 2 . This oxidant produces genotoxic lesions that are repaired by BER, suggesting that the cells have a deficiency in BER affecting survival. In experiments with extracts from the isogenic FEN1 null and wild-type cell lines, the LP-BER activity of FEN1 null cells was deficient, whereas repair by the single-nucleotide BER subpathway was normal. Other consequences of the FEN1 deficiency were also evaluated. These results illustrate that FEN1 plays a role in LP-BER in higher eukaryotes, presumably by processing the flap-containing intermediates of BER. Mol Cancer Res; 8(2); 204-15. ©2010 AACR.

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“…Until recently, when a method for synthesizing oligonucleotides containing C2-AP was reported, little was known about the lesion's biochemical effects (16). Subsequent in vitro experiments revealed that C2-AP in DNA is incised by the Type II E. coli repair enzymes, exonuclease III (Exo III) and endonuclease IV (Endo IV), albeit less efficiently than other abasic lesions (17). Herein we describe the replication of templates containing the C2-AP and the role of SOS polymerases in bypassing the lesion in E. coli using single-stranded shuttle vectors.…”

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Replication of an Oxidized Abasic Site in Escherichia coli by a dNTP-Stabilized Misalignment Mechanism that Reads Upstream and Downstream Nucleotides

et al. 2006

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Abasic sites (AP) and oxidized abasic lesions are often referred to as noninstructive lesions because they cannot participate in Watson-Crick base pairing. The aptness of the term noninstructive for describing AP site replication has been called into question by recent investigations in E. coli using single-stranded shuttle vectors. These studies revealed that the replication of templates containing AP sites or the oxidized abasic lesions resulting from C1′-(L) and C4′-oxidation (C4-AP) are distinct from one another, suggesting that structural features other than Watson-Crick hydrogen bonds contribute to controlling replication. The first description of the replication of the abasic site resulting from formal C2′-oxidation (C2-AP) is presented here. Full-length and single-nucleotide deletion products are observed when templates containing C2-AP are replicated in E. coli. Single nucleotide deletion formation is largely dependent upon the concerted effort of pol II and pol IV, whereas pol V suppresses frameshift product formation. Pol V utilizes the A-rule when bypassing C2-AP. In contrast, pol II and pol IV utilize a dNTP-stabilized misalignment mechanism to read the upstream and downstream nucleotides when bypassing C2-AP. This is the first example in which the identity of the 3′-adjacent nucleotide is read during the replication of a DNA lesion. The results raise further questions as to whether abasic lesions are noninstructive lesions. We suggest that abasic site bypass is affected by the local biopolymer structure in addition to the structure of the lesion.Abasic sites (AP 1 ), the most common DNA lesions formed, are produced in a variety of ways in DNA, including as intermediates during base excision repair (1-3). In addition, a variety of oxidized abasic lesions are produced when DNA is exposed to oxidative stress. DNA damaging agents selectively produce oxidized abasic lesions, including 2-deoxyribonolactone (L), and the C4′-oxidized abasic site (C4-AP) (Figure 1) (4-7). These agents take advantage of the accessibility and/or relatively low bond strength of the respective carbon-hydrogen bonds (8-10). The C2′-carbon-hydrogen bond is the strongest of the deoxyribose bonds in DNA. Consequently, the C2-AP lesion is only produced by highly reactive species, presumably via † Supported by NIGMS grants GM-063028 to M.M.G. and ES-012259 and GM-021422 to M.F.G. * To whom correspondence should addressed. . E-mail: mgreenberg@jhu.edu.. ‡ Johns Hopkins University. § University of Southern California.SUPPORTING INFORMATION AVAILABLE ESI-MS of the oligonucleotides (3) containing C2-AP used to prepare the single-stranded plasmids and a figure describing the REAP experiment. This material is available free of charge via the Internet at http://pubs.acs.org. 1 Abbreviations: C2′-oxidized abasic site, C2-AP; C4′-oxidized abasic site, C4-AP; abasic site, AP; 2-deoxyribonolactone, L; tetrahydrofuran abasic site model, F; polyacrylamide gel electrophoresis, PAGE; DNA polymerase II, pol II; DNA polymerase IV, pol IV; DNA...

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In Vitro Replication and Repair of DNA Containing a C2‘-Oxidized Abasic Site

Cited by 25 publications

References 26 publications

Excision of a Lyase-Resistant Oxidized Abasic Lesion from DNA

Excision of a Lyase-Resistant Oxidized Abasic Lesion from DNA

FEN1 Functions in Long Patch Base Excision Repair Under Conditions of Oxidative Stress in Vertebrate Cells

Replication of an Oxidized Abasic Site in Escherichia coli by a dNTP-Stabilized Misalignment Mechanism that Reads Upstream and Downstream Nucleotides

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