Translesion Replication by DNA Polymerase β Is Modulated by Sequence Context and Stimulated by Fork-like Flap Structures in DNA

Daube, Shirley S.; Arad, Gali; Livneh, Zvi

doi:10.1021/bi991443m

Cited by 15 publications

(8 citation statements)

References 40 publications

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“…2). Moreover, CAG tracts facilitate strand displacement 45 , and the DNA repair polymerase β (Pol β), which can fill in the gap, is stimulated by fork-like flap structures 55 . Normally, a flap generated by BER is removed by flap endonuclease 1 (FEN1), which has 5′ to 3′ exonuclease activity 56-58 (FIG.…”

Section: Large Expansions In Non-dividing Cellsmentioning

confidence: 99%

Mechanisms of trinucleotide repeat instability during human development

2010

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Trinucleotide expansion underlies several human diseases. Expansion occurs during multiple stages of human development in different cell types, and is sensitive to the gender of the parent who transmits the repeats. Repair and replication models for expansions have been described, but we do not know whether the pathway involved is the same under all conditions and for all repeat tract lengths, which differ among diseases. Currently, researchers rely on bacteria, yeast and mice to study expansion, but these models differ substantially from humans. We need now to connect the dots among human genetics, pathway biochemistry and the appropriate model systems to understand the mechanism of expansion as it occurs in human disease.

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Section: Large Expansions In Non-dividing Cellsmentioning

confidence: 99%

Mechanisms of trinucleotide repeat instability during human development

2010

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“…Taken together with the events opposite the abasic site, it appears that TLS at the cDSB occurred via template-directed but sequence-independent polymerization activity. Several mammalian DNA polymerases were shown to be able to bypass an abasic site (38,39,41–44); however, we do not know yet which DNA polymerase carries out the TLS step during the NHEJ events monitored in this study. A potential candidate is DNA polymerase µ, which was implicated to be involved in NHEJ, and was shown by us to possess a template-dependent, but sequence-independent activity during TLS across a synthetic basic site (45).…”

Section: Discussionmentioning

confidence: 91%

Translesion DNA synthesis-assisted non-homologous end-joining of complex double-strand breaks prevents loss of DNA sequences in mammalian cells

Covo

Villartay

Jeggo

et al. 2009

Nucleic Acids Research

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Double strand breaks (DSB) are severe DNA lesions, and if not properly repaired, may lead to cell death or cancer. While there is considerable data on the repair of simple DSB (sDSB) by non-homologous end-joining (NHEJ), little is known about the repair of complex DSBs (cDSB), namely breaks with a nearby modification, which precludes ligation without prior processing. To study the mechanism of cDSB repair we developed a plasmid-based shuttle assay for the repair of a defined site-specific cDSB in cultured mammalian cells. Using this assay we found that repair efficiency and accuracy of a cDSB with an abasic site in a 5′ overhang was reduced compared with a sDSB. Translesion DNA synthesis (TLS) across the abasic site located at the break prevented loss of DNA sequences, but was highly mutagenic also at the template base next to the abasic site. Similar to sDSB repair, cDSB repair was totally dependent on XrccIV, and altered in the absence of Ku80. In contrast, Artemis appears to be specifically involved in cDSB repair. These results may indicate that mammalian cells have a damage control strategy, whereby severe deletions are prevented at the expense of the less deleterious point mutations during NHEJ.

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“…In vitro bypass studies with purified mammalian DNA polymerases have shown that most polymerases can bypass abasic sites, at least under certain conditions (11,24,(50)(51)(52)(53)(54)(55). This underscores the need for in vivo TLR data to determine the physiological role of specific DNA polymerases in bypass of a particular lesion.…”

Section: Discussionmentioning

confidence: 99%

Quantitative measurement of translesion replication in human cells: Evidence for bypass of abasic sites by a replicative DNA polymerase

Avkin

Adar

Blander

et al. 2002

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

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109

104

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Mutations in oncogenes and tumor suppressor genes are critical in the development of cancer. A major pathway for the formation of mutations is the replication of unrepaired DNA lesions. To better understand the mechanism of translesion replication (TLR) in mammals, a quantitative assay for TLR in cultured cells was developed. The assay is based on the transient transfection of cultured cells with a gapped plasmid, carrying a site-specific lesion in the gap region. Filling in of the gap by TLR is assayed in a subsequent bioassay, by the ability of the plasmid extracted from the cells, to transform an Escherichia coli indicator strain. Using this method it was found that TLR through a synthetic abasic site in the adenocarcinoma H1299, the osteogenic sarcoma Saos-2, the prostate carcinoma PC3, and the hepatoma Hep3B cell lines occurred with efficiencies of 92 ؎ 6%, 32 ؎ 2%, 72 ؎ 4%, and 26 ؎ 3%, respectively. DNA sequence analysis showed that 85% of the bypass events in H1299 cells involved insertion of dAMP opposite the synthetic abasic site. Addition of aphidicolin, an inhibitor of DNA polymerases ␣, ␦, and , caused a 4.4-fold inhibition of bypass. Analysis of two XP-V cell lines, defective in DNA polymerase , showed bypass of 89%, indicating that polymerase is not essential for bypass of abasic sites. These results suggest that in human cells bypass of abasic sites does not require the bypass-specific DNA polymerase , but it does require at least one of the replicative DNA polymerases, ␣, ␦, or . The quantitative TLR assay is expected to be useful in the molecular analysis of lesion bypass in a large variety of cultured mammalian cells.T he formation of mutations is a key event in several important biological phenomena, most notably cancer and evolution. A major mechanism for the formation of mutations is the replication of DNA lesions that have escaped DNA repair (1). For many years the molecular mechanism of this process, termed translesion replication (TLR), translesion synthesis, or lesion bypass, was not clear. Recently it was discovered that the main enzyme responsible for carrying out lesion bypass is a novel type of DNA polymerase, specialized for replicating across damaged sites in DNA. Such DNA polymerases were found in organisms ranging from Escherichia coli (DNA polymerase V; refs. 2 and 3) to humans (e.g., DNA polymerase ; refs. 4 and 5). These novel DNA polymerases comprise the new Y superfamily of DNA polymerases (6). Remarkably, humans contain four members of the Y family: DNA polymerases (4, 5), (7,8), and (9-12) and hREV1, which has dCMP transferase activity (13,14). In addition, humans are likely to have an additional polymerase involved in TLR, polymerase , product of the hREV3 and hREV7 genes (refs. 15 and 16; for review see refs. 17-23).Despite the importance of TLR mechanisms in understanding the role of mutagenesis in cancer, and despite the progress made with the discovery of Y family DNA polymerases, little is known about the mechanism of lesion bypass in mammalian cells. This is partl...

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Translesion Replication by DNA Polymerase β Is Modulated by Sequence Context and Stimulated by Fork-like Flap Structures in DNA

Cited by 15 publications

References 40 publications

Mechanisms of trinucleotide repeat instability during human development

Mechanisms of trinucleotide repeat instability during human development

Translesion DNA synthesis-assisted non-homologous end-joining of complex double-strand breaks prevents loss of DNA sequences in mammalian cells

Quantitative measurement of translesion replication in human cells: Evidence for bypass of abasic sites by a replicative DNA polymerase

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