Synthesis by DNA polymerase I on bleomycin-treated deoxyribonucleic acid: a requirement for exonuclease III

Niwa, Osami; Moses, Robb E.

doi:10.1021/bi00505a002

Cited by 35 publications

(21 citation statements)

References 21 publications

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“…Thus, the 5' sugar-phosphate linkage is apparently more labile in the NCS-induced site than in the authentic AP site. Other agents, including ionizing radiation and bleomycin, also produce oxidized AP sites (26,27) that reportedly show altered susceptibilities to certain AP endonucleases (28)(29)(30).…”

Section: Resultsmentioning

confidence: 99%

Endonuclease-resistant apyrimidinic sites formed by neocarzinostatin at cytosine residues in DNA: evidence for a possible role in mutagenesis.

Povirk¹,

Goldberg²

1985

Proc. Natl. Acad. Sci. U.S.A.

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When defined-sequence DNA from the lacI region of plasmid pMC1 was treated with the nonprotein chromophore of neocarzinostatin in the presence of various thiols, the predominant lesions were direct strand breaks, occurring primarily at thymine and adenine residues. In the presence of glutathione, however, alkali-dependent strand breaks, occurring at certain cytosine residues, were also detected but were virtually absent when other thiols were used. Chromophore-induced release of free cytosine base from[3H]cytosine-labeled DNA was 2-to 3-fold greater with glutathione than with the other thiols. These results suggest that the alkali-dependent strand break is some form of apyrimidinic site. These sites were substrates for endonuclease IV of Escherichia coli, although a 5-fold greater concentration of enzyme was required for their cleavage than was required for cleavage of apurinic sites in depurinated DNA. These sites were also less sensitive to E. coli endonuclease VI (exonuclease III) by a factor of at least 5 and less sensitive to E. coli endonuclease III by a factor of at least 10. These and other results suggest that these sites are chemically different from normal apurinic/apyrimidinic sites. When chromophore-induced apyrimidinic sites were quantitated as alkali-dependent breaks at 11 specific sites in the lacI gene, a correlation was found between occurrences of these lesions and the reported frequencies of G-C to A-T transitions at the same sites. All occurrences of the trinucleotide sequence A-G-C, including the ochre 21 mutational hot spot, were particularly prominent sites. The selective formation of endonuclease-resistant apyrimidinic sites at specific cytosine residues may explain the high frequency of G-C to A-T transitions in the mutational spectrum of neocarzinostatin.Studies of forward mutations induced in the lacI gene of Escherichia coli by various agents have strongly suggested that mutations are targeted at sites of DNA damage (1-3). Neocarzinostatin (NCS) is an SOS-dependent mutagen that induces base substitutions and, to a lesser extent, frameshifts (4). Studies of NCS-induced mutations in the lacI gene (5) revealed that all types of monitorable base substitutions were induced, with some preference for G-C to A-T transitions (A-T to G-C transitions are not monitorable in this system). The spectrum was dominated by a G-C to A-T transition hot spot at the ochre 21 locus. Several less prominent hot spots were also present, whereas no mutations at all were detected at nearly half the monitorable sites. Since the predominant lesions induced in DNA by NCS (6, 7) or its isolated nonprotein chromophore (8) are strand breaks occurring mainly at thymine and adenine residues, it seems likely that many of the mutations, particularly those occurring at G-C base pairs, result from other, minor lesions, which are formed with a different base specificity.Since NCS releases free bases from DNA (9, 10), it is possible that apurinic/apyrimidinic (AP) sites with an intact sugar-phosphate backbone are for...

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Section: Resultsmentioning

confidence: 99%

Endonuclease-resistant apyrimidinic sites formed by neocarzinostatin at cytosine residues in DNA: evidence for a possible role in mutagenesis.

Povirk¹,

Goldberg²

1985

Proc. Natl. Acad. Sci. U.S.A.

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“…Hence, at least some types of BLM damage do not serve as suitable primer termini for DNA polymerase I activity. Indeed, Niwa & Moses (1981) have shown that BLM-damaged DNA is not a suitable substrate for purified DNA polymerase I activity in vitro. However, purified E. coli exonuclease I11 is able to convert BLM-damaged DNA into a suitable template-primer for polymerization (Niwa & Moses,198 1).…”

Section: Discussionmentioning

confidence: 99%

Plasmid R46-mediated Protection Against Bleomycin is polA+-dependent

Attfield

Pinney

1982

Microbiology

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Strains of Escherichia coli deficient in post-replication recombination repair were more sensitive to bleomycin than wild-type, repair-proficient strains. Mutants lacking excision repair functions were no more sensitive to bleomycin than the wild-type strains, indicating that this pathway is not involved in the repair of bleomycin-damaged DNA. Plasmid R46 not only protected repair-proficient strains but also those with recB, re&, uvrA or lig genotypes, suggesting that R46 protection against bleomycin is independent of these host repair functions. However, R46 protection was abolished in recA or polA strains, indicating that these gene functions are necessary for plasmid-mediated protection. It is suggested that protection may be due to a recA+-dependent interaction of a plasmid-encoded product with host DNA polymerase I, resulting in an increase in the DNA repair capacity of cells.

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“…By contrast, adducts induced by small alkylating agents are recognized by DNA glycosylases that remove the damaged base, leaving apurinic/apyrimidinic (AP) sites that are cleaved by AP endonucleases (3, 7). y Radiation produces strand breaks with nonligatable 3' ends, requiring the action of a 3' --5' exonuclease for repair (8,9).Much of our knowledge of uvrABC-mediated DNA repair is based on studies using cell-free systems. Recently an in vitro excision repair system using human cell-free extracts (CFEs) has been described (10, 11).…”

mentioning

confidence: 99%

Repair of DNA damaged by UV light and ionizing radiation by cell-free extracts prepared from Schizosaccharomyces pombe.

Sidik

Lieberman

Freyer

1992

Proc. Natl. Acad. Sci. U.S.A.

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A whole cell extract prepared from Schizosaccharomyces pombe was shown to be active in an assay for repair of plasmid DNA damaged by either ultraviolet (IV) light or vrradiation. The assay allows for analysis of repair synthesis at single-strand nicks generated by vrays and analysis of the incision step and repair synthesis in UV-ilghtdamaged DNA. Repair synthesis ofDNA damagedbyeitherUV light or v-rays was shown to depend on the presence of AT? in the reaction mixture. However, incision at pyrimdhne dimers did not require the addi of exogenous ATP. These studies showed that p Masmd DNA containing a single pi e dimer or one single-strand nick is a suitable substrate in this assay system. S. pombe is a geneticall well-defined eukaryotic organism and many radiation-senstive mutant derivatives have already been described, m ng this a powerful system In which to study DNA excision repair.Perhaps the best-studied DNA repair system is the excision repair pathway (reviews, refs. 1-3). Excision repair is a "cut and patch" mechanism, whereby the damaged bases and adjacent nucleotides are removed and new bases are synthesized in a template-dependent reaction. This is the mechanism by which much ofthe DNA damage caused by UV light, alkylating agents and ionizing radiation is repaired. Much of our knowledge about excision repair has come from studies of the Escherichia coli uvrABC system, involved in the repair of DNA containing pyrimidine dimers generated by UV radiation (4). This multiprotein complex binds DNA at a site distant from the damage and travels to the damaged site via an associated ATPase activity (5). This complex cleaves the DNA exactly eight bases on the 5' side of the pyrimidine dimer and four or five bases to the 3' side (6}. The damagecontaining oligonucleotide is removed by the complex, and repair synthesis then follows (4). By contrast, adducts induced by small alkylating agents are recognized by DNA glycosylases that remove the damaged base, leaving apurinic/apyrimidinic (AP) sites that are cleaved by AP endonucleases (3, 7). y Radiation produces strand breaks with nonligatable 3' ends, requiring the action of a 3' --5' exonuclease for repair (8,9).Much of our knowledge of uvrABC-mediated DNA repair is based on studies using cell-free systems. Recently an in vitro excision repair system using human cell-free extracts (CFEs) has been described (10, 11). These extracts are active in repairing UV-damaged DNA as well as DNA treated with cisplatin and psoralen (10)(11)(12). A requirement for human single-strand binding protein in repair of UV-damaged DNA was demonstrated, along with a requirement for proliferating cell nuclear antigen (13,14). The patch size generated during the repair of pyrimidine dimers was found to be 27-29 nucleotides long (15). Single-strand breaks generated by t-rays were shown to be repaired in human CFE in a (3-NAD+-dependent reaction, which is believed to be mediated through the action ofpoly(ADP-ribose) polymerase (16).Here we report the development of an in vitro DNA ...

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Synthesis by DNA polymerase I on bleomycin-treated deoxyribonucleic acid: a requirement for exonuclease III

Cited by 35 publications

References 21 publications

Endonuclease-resistant apyrimidinic sites formed by neocarzinostatin at cytosine residues in DNA: evidence for a possible role in mutagenesis.

Endonuclease-resistant apyrimidinic sites formed by neocarzinostatin at cytosine residues in DNA: evidence for a possible role in mutagenesis.

Plasmid R46-mediated Protection Against Bleomycin is polA+-dependent

Repair of DNA damaged by UV light and ionizing radiation by cell-free extracts prepared from Schizosaccharomyces pombe.

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