Two-polymerase mechanisms dictate error-free and error-prone translesion DNA synthesis in mammals

Shachar, Sigal; Ziv, Omer; Avkin, Sharon; Adar, Sheera; Wittschieben, John P.; Reiner, Thomas; Chaney, Stephen G.; Friedberg, Errol C.; Wang, Zhigang; Carell, Thomas; Geacintov, Nicholas E.; Livneh, Zvi

doi:10.1038/emboj.2009.72

Cited by 73 publications

(135 citation statements)

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“…We similarly assayed two additional DNA lesions: (i) a TT 6-4 PP adduct, representing one of the two major UV light-induced DNA lesions [unlike BP-G, this is not a bulky lesion, but it distorts DNA (1)], and (ii) an artificial and extreme type of lesion, a trimethylene [(CH 2 ) 3 ] insert into the DNA backbone. We have previously shown, using a gapped plasmid TLS assay, that this hydrocarbon non-DNA insert, termed trimethylene (M3), can be tolerated by TLS both in E. coli (29) and in human cells (28,30). As can be seen in Fig.…”

Section: Resultsmentioning

confidence: 90%

“…To discriminate between the two tolerance mechanisms we engineered opposite the lesions mismatched nucleotides, which are infrequently incorporated during TLS. For example, on the basis of plasmid assays, the DNA adduct benzo[a]pyrene-guanine (BP-G), which is formed by tobacco smoke, is bypassed relatively accurately in human cells (∼90% insertion of the correct C), with the main mutagenic event being misinsertion of an A, which together account for 94% of all TLS events (28). Thus, when constructing lesion shuttle vectors with BP-G lesions (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, when constructing lesion shuttle vectors with BP-G lesions (Fig. 3B), we engineered opposite the lesions T and/or G, which are infrequently inserted by TLS (2% and 4%, respectively) (28). In this configuration, the sequence obtained after tolerating the lesions and cell propagation for multiple generations is indicative of the tolerance mechanism and enables discrimination between accurate TLS, mutagenic TLS, and HDR (Fig.…”

Section: Resultsmentioning

confidence: 99%

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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: Resultsmentioning

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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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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“…Several lines of evidence indicate that the mutagenic bypass of CTDs occur in a Polz-dependent manner if Polh is unavailable (Gibbs et al, 2005;Shachar et al, 2009). We suppose that the increased mutation frequency in polh-1 was caused by AtPolz (and AtRev1), which complements AtPolh for the bypass of CTDs (Fig.…”

Section: Mutation Frequency In Rev1 Rev3 Double Mutantsmentioning

confidence: 92%

Role of AtPolζ, AtRev1, and AtPolη in UV Light-Induced Mutagenesis in Arabidopsis

et al. 2010

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Translesion synthesis (TLS) is a DNA damage tolerance mechanism in which DNA lesions are bypassed by specific polymerases. To investigate the role of TLS activities in ultraviolet light-induced somatic mutations, we analyzed Arabidopsis (Arabidopsis thaliana) disruptants of AtREV3, AtREV1, and/or AtPOLH genes that encode TLS-type polymerases. The mutation frequency in rev3-1 or rev1-1 mutants decreased compared with that in the wild type, suggesting that AtPolz and AtRev1 perform mutagenic bypass events, whereas the mutation frequency in the polh-1 mutant increased, suggesting that AtPolh performs nonmutagenic bypass events with respect to ultraviolet light-induced lesions. The rev3-1 rev1-1 double mutant showed almost the same mutation frequency as the rev1-1 single mutant. The increased mutation frequency found in polh-1 was completely suppressed in the rev3-1 polh-1 double mutant, indicating that AtPolz is responsible for the increased mutations found in polh-1. In summary, these results suggest that AtPolz and AtRev1 are involved in the same (error-prone) TLS pathway that is independent from the other (error-free) TLS pathway mediated by AtPolh.

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“…Após o bypass, PCNA é desubiquitinado pela DUB USP1, o que leva à perda da afinidade da pol TLS e à volta da pol replicativa (Huang et al, 2006)(Figura 7). Dependendo do tipo de lesão encontrado, como por exemplo adutos de cisplatina ou sítios AP, o processo de bypass pode depender da ação sequencial de duas polimerases, uma responsável pela inserção do nucleotídeo frente à lesão e outra pela extensão do novo fragmento de DNA (Shachar et al, 2009). …”

Section: Mecanismos De Tolerância a Dano: Síntese Translesão (Tls)unclassified

Papel das proteínas XPD e DNA polimerase eta nas respostas de células humanas a danos no genoma

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Two-polymerase mechanisms dictate error-free and error-prone translesion DNA synthesis in mammals

Cited by 73 publications

References 15 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

Role of AtPolζ, AtRev1, and AtPolη in UV Light-Induced Mutagenesis in Arabidopsis

Papel das proteínas XPD e DNA polimerase eta nas respostas de células humanas a danos no genoma

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