Analysis of Unassisted Translesion Replication by the DNA Polymerase III Holoenzyme

Tomer, Guy; Livneh, Zvi

doi:10.1021/bi982599+

Cited by 31 publications

(32 citation statements)

References 52 publications

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“…Plasmid pLSV5 was constructed in four steps. First, the puromycin resistance gene under the phosphoglycerate kinase (PGK) promoter was cloned into the kanamycin-carrying plasmid pSKSL (53) to generate the plasmid pSKSL-puro. The puromycin resistance gene was obtained by PCR from pRetrosuper, using the following primers: 5′-GGTAGGGAAT-TCGCTTTTCCAAGGCAGTC-3′ and 5′-GATGCATGGGATCCTGCGCTCCTTTCGG-3′.…”

Section: Methodsmentioning

confidence: 99%

Genomic assay reveals tolerance of DNA damage by both translesion DNA synthesis and homology-dependent repair in mammalian cells

Izhar

Ziv

Cohen

et al. 2013

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

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DNA lesions can block replication forks and lead to the formation of single-stranded gaps. These replication complications are mitigated by DNA damage tolerance mechanisms, which prevent deleterious outcomes such as cell death, genomic instability, and carcinogenesis. The two main tolerance strategies are translesion DNA synthesis (TLS), in which low-fidelity DNA polymerases bypass the blocking lesion, and homology-dependent repair (HDR; postreplication repair), which is based on the homologous sister chromatid. Here we describe a unique high-resolution method for the simultaneous analysis of TLS and HDR across defined DNA lesions in mammalian genomes. The method is based on insertion of plasmids carrying defined site-specific DNA lesions into mammalian chromosomes, using phage integrase-mediated integration. Using this method we show that mammalian cells use HDR to tolerate DNA damage in their genome. Moreover, analysis of the tolerance of the UV light-induced 6-4 photoproduct, the tobacco smokeinduced benzo[a]pyrene-guanine adduct, and an artificial trimethylene insert shows that each of these three lesions is tolerated by both TLS and HDR. We also determined the specificity of nucleotide insertion opposite these lesions during TLS in human genomes. This unique method will be useful in elucidating the mechanism of DNA damage tolerance in mammalian chromosomes and their connection to pathological processes such as carcinogenesis.error-prone DNA repair | homologous recombination repair | recombinational repair D NA damage is abundant, caused by both external agents such as sunlight and tobacco smoke and intracellular byproducts of metabolism, amounting to about 50,000 lesions per day per cell (1). Despite the presence of effective DNA repair mechanisms that eliminate lesions and restore the original DNA sequence, DNA replication often encounters unrepaired lesions that have escaped repair. These DNA damages may cause arrest of replication forks and the generation of postreplication gaps (2, 3). To complete replication and prevent the formation of double-strand breaks, which are highly deleterious, cells use DNA damage tolerance (DDT) mechanisms. These include translesion DNA synthesis (TLS) and homology-dependent repair (HDR), which enable bypass of the lesions and completion of replication, without removing the lesions from DNA. HDR uses the sequence from the intact sister chromatid to patch the single-stranded template region carrying the lesion. [We term HDR the pathways of DNA damage tolerance that rely on the homologous sister chromatid, also termed postreplication repair (PRR), damage avoidance, template switch, copy choice recombination, and homologous recombination repair.] This is carried out either by physical transfer of the segment complementary to the damaged template [also termed homologous recombination repair (HRR)] or by copying the complementary strand from the sister chromatid (template switch or postreplication repair). TLS employs specialized low-fidelity DNA polymerases to replicate across ...

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Section: Methodsmentioning

confidence: 99%

Genomic assay reveals tolerance of DNA damage by both translesion DNA synthesis and homology-dependent repair in mammalian cells

Izhar

Ziv

Cohen

et al. 2013

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

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“…The construction of the control gapped plasmid is described in ref. 45. A gapped plasmid with a site-specific TT CPD in the sequence context: 5Ј-GCAAGTTGGAG-3Ј (the CPD is underlined) was constructed as described in ref.…”

Section: Methodsmentioning

confidence: 99%

DNA polymerase ζ cooperates with polymerases κ and ι in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients

Ziv

Geacintov

Nakajima

et al. 2009

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

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117

125

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Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase , the POLH gene product. A deficiency in DNA polymerase due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase cooperates with DNA polymerases and to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases and , but not , protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load.carcinogenesis ͉ DNA repair ͉ lesion bypass ͉ replication ͉ ultraviolet T LS is a fundamental mechanism for tolerating DNA damage that has escaped repair, carried out by specialized lowfidelity DNA polymerases, which synthesize across a wide variety of DNA lesions (1). At least 5 TLS DNA polymerases are present in mammals, four of which, DNA polymerases , , , and REV1, belong to the Y superfamily. The fifth TLS polymerase is pol, which belongs to the B family, and is the only TLS polymerase known to be essential in mammals (2-5). TLS polymerases exhibit a certain degree of specificity for their substrate DNA lesions, and their activity is tightly regulated (6-9). The most well characterized TLS polymerase is pol, which is specialized to bypass cyclobutane pyrimidine dimers (CPD) in a relatively error-free manner. The biological significance of pol is illustrated by the hereditary disease xeroderma pigmentosum variant (XPV), in which germ-line mutations in the POLH gene, encoding pol, cause an extreme 1000-fold increased predisposition to sunlight-induced skin cancer (10, 11). Cells from XPV patients exhibit a slightly increased UV sensitivity, and a dramatic UV hypermutability (12), which is responsible for their extreme cancer predisposition. The UV hypermutability is explained by the activity of a back-up DNA polymerase that performs TLS across CPD with lower efficiency and higher error-frequency. Although there is evidence that pol is involved in TLS across CPD in XPV cells (13-15), additional polymerases may be involved, and the picture is far from being complete. This is an important issue because these polymerases are likely to be driving sunlight-induced skin carcinogenesis in XPV patients.Here, we show that 3 TLS polymerases, pol, pol, and pol, are involved in TLS across CPD in XPV cells. Moreover, pol and pol, but not pol, also provide protection against UV cytotoxicity, independently of nucleotide excision repair (NER). Results Pol, pol, and pol Are Involved in T...

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“…The construction of the gap-lesion plasmids GP21 (with a synthetic abasic site) and GP20 (without a lesion; Fig. 1B) was previously described (29,30). The gap-lesion plasmids GP-BPG1 and GP-BPG2 were constructed similarly to GP21, except that the insert oligonucleotides contained the BPDE-modified 54-mer templates (Fig.…”

Section: Methodsmentioning

confidence: 99%

Quantitative Analysis of Translesion DNA Synthesis across a Benzo[a]pyrene-Guanine Adduct in Mammalian Cells

Avkin

Goldsmith

Velasco-Miguel

et al. 2004

Journal of Biological Chemistry

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175

177

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Replication across unrepaired DNA lesions in mammalian cells is effected primarily by specialized, low fidelity DNA polymerases. We studied translesion DNA synthesis (TLS) across a benzo[a]pyrene-guanine (BP-G) adduct, a major mutagenic DNA lesion generated by tobacco smoke. This was done using a quantitative assay that measures TLS indirectly, by measuring the recovery of gapped plasmids transfected into cultured mammalian cells. Analysis of PolK ؉/؉ mouse embryo fibroblasts (MEFs) showed that TLS across the BP-G adduct occurred with an efficiency of 48 ؎ 4%, which is an order of magnitude higher than in Escherichia coli. In PolK ؊/؊ MEFs, bypass was 16 ؎ 1%, suggesting that at least twothirds of the BP-G adducts in MEFs were bypassed exclusively by polymerase (pol ). In contrast, pol was not required for bypass across BP-G in a human XP-V cell line. Analysis of misinsertion specificity across BP-G revealed that bypass was more error-prone in MEFs lacking pol . Expression of pol from a plasmid introduced into PolK ؊/؊ MEFs restored both the extent and fidelity of bypass across BP-G. Pol was not required for bypass of a synthetic abasic site. In vitro analysis demonstrated efficient bypass across BP-G by both pol and pol , suggesting that the biological role of pol in TLS across BP-G is due to regulation of TLS and not due to an exclusive ability to bypass this lesion. These results indicate that BP-G is bypassed in mammalian cells with relatively high efficiency and that pol bypasses BP-G in vivo with higher efficiency and higher accuracy than other DNA polymerases.Genomic DNA is constantly subject to damage caused by both external agents, such as sunlight, and endogenous chemicals, such as reactive oxygen species. Most of this damage is eliminated by error-free DNA repair mechanisms, thereby restoring the DNA to its native sequence (1). However, a significant number of lesions escape repair and might therefore interfere with DNA replication and gene expression. Such interference can be mitigated by DNA damage tolerance mechanisms, primarily translesion DNA synthesis (TLS 1 ; also termed translesion replication) (2-5) and postreplicative recombinational repair (1, 6 -8). The key components in TLS are low fidelity DNA polymerases that specialize in lesion bypass (9 -12). These proteins were conserved in evolution and are present in organisms ranging from Escherichia coli to humans (13). Humans contain at least four specialized DNA polymerases belonging to the Y superfamily (pol , pol , pol , and REV1) as well as several from other polymerase families (e.g. pol (14), pol (15, 16), and pol (16, 17)). Many of these polymerases have been implicated in TLS in vitro (18 -24). However, there is a paucity of information about the efficiency and fidelity with which they support lesion bypass in living cells.Pol has a well established biological role in TLS, since it is mutated in all patients examined with the variant form of the hereditary disease xeroderma pigmentosum (10,18). This disease is characterized by sensitivi...

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Analysis of Unassisted Translesion Replication by the DNA Polymerase III Holoenzyme

Cited by 31 publications

References 52 publications

Genomic assay reveals tolerance of DNA damage by both translesion DNA synthesis and homology-dependent repair in mammalian cells

Genomic assay reveals tolerance of DNA damage by both translesion DNA synthesis and homology-dependent repair in mammalian cells

DNA polymerase ζ cooperates with polymerases κ and ι in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients

Quantitative Analysis of Translesion DNA Synthesis across a Benzo[a]pyrene-Guanine Adduct in Mammalian Cells

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