DNA Polymerase I-Mediated Translesion Synthesis in RecA-Independent DNA Interstrand Cross-Link Repair in <i>E. coli</i>

Zietlow, Laura; Bessho, Tadayoshi

doi:10.1021/bi702343y

Cited by 7 publications

(16 citation statements)

References 21 publications

(33 reference statements)

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“…We prepared a 53-nt length template DNA with a 12-mer oligonucleotide attached via psoralen ICL and placed a four-nucleotide gap between a primer end and a 5’-end of an unhooked strand (Figure 2). Similar types of substrate DNA have been used to study the TLS activity of an ICL 9, 13, 14, 21 . A primer-extension to the attached oligonucleotide by a DNA polymerase results in the production of a 26-nt length fragment.…”

Section: Resultsmentioning

confidence: 99%

Bypass of a Psoralen DNA Interstrand Cross-Link by DNA Polymerases β, ι, and κ in Vitro

et al. 2012

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Repair of DNA inter-strand cross-links in mammalian cells involves several biochemically distinctive processes, including the release of one of the cross-linked strands and translesion DNA synthesis (TLS). In this report, we investigated in vitro TLS activity of psoralen DNA inter-strand cross-link by three DNA repair polymerases, DNA polymerase beta, kappa and iota. DNA polymerase beta is capable of bypassing a psoralen cross-link with a low efficiency. Cell extracts prepared from DNA polymerase beta knockout mouse embryonic fibroblast showed a reduced bypass activity of the psoralen cross-link and purified DNA polymerase beta restored the bypass activity. In addition, DNA polymerase iota mis-incorporated thymine across the psoralen cross-link and DNA polymerase kappa extended these mis-paired primer ends, suggesting that DNA polymerase iota may serve as an inserter and DNA polymerase kappa may play a role as an extender in the repair of psoralen DNA inter-strand cross-links. The results demonstrated here indicate that multiple DNA polymerases could participate in TLS steps in mammalian DNA inter-strand cross-link repair.

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Section: Resultsmentioning

confidence: 99%

Bypass of a Psoralen DNA Interstrand Cross-Link by DNA Polymerases β, ι, and κ in Vitro

et al. 2012

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“…3Aand 3B) (23). The 74-mer/12-mer was hybridized to a complementary upstream 36-mer or 22-mer, to generate either a nicked or gapped DNA substrate (23). …”

Section: Resultsmentioning

confidence: 99%

“…Under these conditions, the cross-linked substrate is well separated from the non-cross-linked substrate. Typically, less than 0.3 % of the substrate was non-cross-linked (23). For the undamaged substrate, a 32 P-labeled 22-mer primer was annealed to the 74-mer template, or a 32 P- labeled 18-mer primer was annealed to a 31-mer template.…”

Section: Methodsmentioning

confidence: 99%

Evidence for the Involvement of Human DNA Polymerase N in the Repair of DNA Interstrand Cross-Links

et al. 2009

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Human DNA polymerase N (PolN) is an A-family nuclear DNA polymerase whose function is unknown. This study examines the possible role of PolN in DNA repair in human cells treated with PolN-targeted siRNA. HeLa cells with siRNA-mediated knockdown of PolN were more sensitive than control cells to DNA cross-linking agent mitomycin C (MMC), but were not hyper-sensitive to UV irradiation. The MMC hyper-sensitivity of PolN knockdown cells was rescued by the overexpression of DNA polymerase-proficient PolN but not by DNA polymerase-deficient PolN. Furthermore, in vitro experiments showed that purified PolN conducts low efficiency non-mutagenic bypass of a psoralen DNA interstrand cross-link (ICL), whose structure resembles an intermediate in the proposed pathway of ICL repair. These results suggest that PolN might play a role in translesion DNA synthesis during ICL repair in human cells. KeywordsDNA interstrand cross-links; DNA polymerase N; TLS; Psoralen; Chemotherapeutics E. coli PolI is a high fidelity DNA repair polymerase that conducts gap-filling DNA synthesis during nucleotide excision repair (NER), base excision repair (BER), and DNA interstrand cross-link (ICL) repair. E. coli PolI is the prototypical member of A-family DNA polymerases (1). Drosophila melanogaster Mus308 is an A-family nuclear DNA polymerase, the mutants of which are hyper-sensitive to DNA cross-linking agents but not to other DNA damaging agents (2)(3)(4). This suggests that Mus308 may play a role in ICL repair in Drosophila. Two nuclear A-family DNA polymerases, DNA polymerase N (PolN) and DNA polymerase Q (PolQ) were recently discovered (5,6). It has been proposed that PolN and PolQ are mammalian orthologs of Mus308 and that they participate in ICL repair in mammalian cells. However, additional studies are needed to confirm the precise role(s) of PolN and PolQ in DNA repair in mammalian cells.ICLs are generated endogenously as bi-functional products of lipid peroxidation and by exogenous exposure to DNA damaging agents, some of which are commonly used as cancer chemotherapeutic drugs (7,8). Because ICLs covalently link the two complementary strands of duplex DNA, they prevent progression of the DNA replication fork and block RNA transcription. This property makes ICLs highly toxic to proliferating cells. The molecular mechanism of human ICL repair is poorly understood (7-10). The current model of mammalian ICL repair (7-10) suggests that when a DNA replication fork stalls at an ICL, it is recognized *To whom correspondence should be addressed. tbessho@unmc by an endonuclease that generates a double-strand break (DSB) in the vicinity of the ICL (11)(12)(13)(14). Subsequently, XPF-ERCC1 unhooks the ICL, resulting in a gap across from the unhooked ICL (14-17), which cannot be sealed by replicative DNA polymerases δ or ε. Gaps generated during ICL repair are thought to be repaired by translesion DNA synthesis (TLS) or by homologous recombination using the homologous chromosome (the sister chromatid is not available). Once the gap is sea...

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“…Zietlow and Bessho [2008] showed that Pol I was able to bypass a psoralen ICL, preferentially incorporating a dAMP opposite the adducted thymine. This bypass was however very inefficient (below 1%), and it remains to be determined if the reaction could rendered more efficient by reducing the amount of dsDNA around the ICL.…”

Section: Pol I Pol Ii and Pol Iv Have Been Implicated In Icl Repairmentioning

confidence: 99%

Translesion DNA synthesis polymerases in DNA interstrand crosslink repair

Schärer

2010

Environ and Mol Mutagen

112

120

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DNA interstrand crosslinks (ICLs) are induced by a number of bifunctional antitumor drugs such as cisplatin, mitomycin C, or the nitrogen mustards as well as endogenous agents formed by lipid peroxidation. The repair of ICLs requires the coordinated interplay of a number of genome maintenance pathways, leading to the removal of ICLs through at least two distinct mechanisms. The major pathway of ICL repair is dependent on replication, homologous recombination, and the Fanconi anemia (FA) pathway, whereas a minor, G0/G1-specific and recombination-independent pathway depends on nucleotide excision repair. A central step in both pathways in vertebrates is translesion synthesis (TLS) and mutants in the TLS polymerases Rev1 and Pol zeta are exquisitely sensitive to crosslinking agents. Here, we review the involvement of Rev1 and Pol zeta as well as additional TLS polymerases, in particular, Pol eta, Pol kappa, Pol iota, and Pol nu, in ICL repair. Biochemical studies suggest that multiple TLS polymerases have the ability to bypass ICLs and that the extent ofbypass depends upon the structure as well as the extent of endo- or exonucleolytic processing of the ICL. As has been observed for lesions that affect only one strand of DNA, TLS polymerases are recruited by ubiquitinated proliferating nuclear antigen (PCNA) to repair ICLs in the G0/G1 pathway. By contrast, this data suggest that a different mechanism involving the FA pathway is operative in coordinating TLS in the context of replication-dependent ICL repair.

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DNA Polymerase I-Mediated Translesion Synthesis in RecA-Independent DNA Interstrand Cross-Link Repair in E. coli

Cited by 7 publications

References 21 publications

Bypass of a Psoralen DNA Interstrand Cross-Link by DNA Polymerases β, ι, and κ in Vitro

Bypass of a Psoralen DNA Interstrand Cross-Link by DNA Polymerases β, ι, and κ in Vitro

Evidence for the Involvement of Human DNA Polymerase N in the Repair of DNA Interstrand Cross-Links

Translesion DNA synthesis polymerases in DNA interstrand crosslink repair

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