Replication Bypass of Interstrand Cross-link Intermediates by Escherichia coli DNA Polymerase IV

Kumari, Anuradha; Minko, Irina G.; Harbut, Michael B.; Finkel, Steven E.; Goodman, Myron F.; Lloyd, R. Stephen

doi:10.1074/jbc.m801237200

Cited by 49 publications

(72 citation statements)

References 21 publications

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“…‫,ء‬ P Ͻ 0.01; ‫,ءء‬ P Ͻ 0.005. ϩ and Ϫ denote growth or lack of growth of AH109 recombinants on LTH dropout-supplemented media. NQO) (27,34,40,43,54,56,67). Loss of dinB also sensitized E. coli to the cytotoxic effects of alkylating agents (4).…”

Section: Resultsmentioning

confidence: 99%

“…The association of Y family polymerases with inducible mutagenesis has implicated these enzymes in the adaptation of bacteria to environmental stress (17,20,39,54,58,59,66). Their key properties are exemplified in E. coli Pol IV: the polymerase catalyzes efficient and accurate TLS across certain N 2 -dG adducts (27,28,34,40,45,67) and has been implicated in the tolerance of alkylation damage (4); furthermore, overexpression of Pol IV significantly increases mutation rates in E. coli (reviewed in references 21 and 26), and dinB is the only SOS-regulated gene required at induced levels for stress-induced mutagenesis in this organism (20). Furthermore, overproduction of E. coli Pol IV inhibits replication fork progression through replacement of the replicative polymerase to form an alternate replisome in which Pol IV modulates the rate of unwinding of the DnaB helicase (25) and also reduces colony-forming ability (61).…”

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Role of the DinB Homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis

et al. 2010

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The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phasedependent manner, with dinB2 levels 12-to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the ␤-clamp, consistent with its canonical C-terminal ␤-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N 2 -dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.The emergence and global spread of multi-and extensively drug-resistant strains of Mycobacterium tuberculosis have further complicated the already daunting challenge of controlling tuberculosis (TB) (15). The mechanisms that underlie the evolution of drug resistance in M. tuberculosis by chromosomal mutagenesis and their association with the conditions that tubercle bacilli encounter during the course of infection are poorly understood (6). It has been postulated that hypoxia, low pH, nutrient deprivation, and nitrosative and oxidative stress impose environmental and host immune-mediated DNA-damaging insults on infecting bacilli (64). In addition, the observed importance of excision repair pathways for the growth and survival of M. tuberculosis in murine models of infection (13, 55) and the upregulation of M. tuberculosis genes involved in DNA repair and modification in pulmonary TB in humans provide compelling evidence that the in vivo environment is DNA damaging (51).Damage tolerance constitutes an integral component of an organism's response to genotoxic stress, preventing collapse of the replication fork at persisting, replication-blocking lesions through the engagement of specialized DNA polymerases that are able to catalyze translesion synthesis (TLS) across the sites of damage (19,21,60). Most TLS polymerases belong to the Y family, which comprises a wide range of structurally related proteins present in bacteria, archaea, and eukaryotes (44). Of these, the DinB subfamily of Y family po...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Role of the DinB Homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis

et al. 2010

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“…ΔdinB strains are sensitive to DNA-damaging agents, nitrofurazone (NFZ) and 4-nitroquinolone-1-oxide (4-NQO), and DinB preferentially and accurately bypasses a structural analog of the major NFZ-induced N 2 -dG lesion as well as certain other N 2 -dG adducts (10)(11)(12)(13). NusA is an essential, multidomain protein that functions in both termination and antitermination of transcription, and is associated with the RNA polymerase (RNAP) throughout the elongation and termination phases of transcription (14)(15)(16)(17)(18)(19)(20)(21)(22).…”

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Roles for the transcription elongation factor NusA in both DNA repair and damage tolerance pathways in Escherichia coli

Cohen

Lewis

Mooney³

et al. 2010

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

113

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We report observations suggesting that the transcription elongation factor NusA promotes a previously unrecognized class of transcription-coupled repair (TCR) in addition to its previously proposed role in recruiting translesion synthesis (TLS) DNA polymerases to gaps encountered during transcription. Earlier, we reported that NusA physically and genetically interacts with the TLS DNA polymerase DinB (DNA pol IV). We find that Escherichia coli nusA11(ts) mutant strains, at the permissive temperature, are highly sensitive to nitrofurazone (NFZ) and 4-nitroquinolone-1-oxide but not to UV radiation. Gene expression profiling suggests that this sensitivity is unlikely to be due to an indirect effect on gene expression affecting a known DNA repair or damage tolerance pathway. We demonstrate that an N 2 -furfuryl-dG (N 2 -f-dG) lesion, a structural analog of the principal lesion generated by NFZ, blocks transcription by E. coli RNA polymerase (RNAP) when present in the transcribed strand, but not when present in the nontranscribed strand. Our genetic analysis suggests that NusA participates in a nucleotide excision repair (NER)-dependent process to promote NFZ resistance. We provide evidence that transcription plays a role in the repair of NFZinduced lesions through the isolation of RNAP mutants that display altered ability to survive NFZ exposure. We propose that NusA participates in an alternative class of TCR involved in the identification and removal of a class of lesion, such as the N 2 -f-dG lesion, which are accurately and efficiently bypassed by DinB in addition to recruiting DinB for TLS at gaps encountered by RNAP.T he process of nucleotide excision repair (NER) acts to remove a wide variety of DNA lesions and in Escherichia coli is mediated through the concerted action of the uvrA, uvrB, and uvrC gene products (1). The process of transcription-coupled repair (TCR) targets NER to actively transcribed genes, resulting in preferential repair of the transcribed strand relative to the nontranscribed strand (2-4). In E. coli, the mfd + gene product couples the process of NER to transcription, and has been shown to be responsible for the strand specific repair of UV-induced lesions (5-7).We have recently reported that the highly conserved TLS polymerase DinB (DNA pol IV), a member of the class of specialized DNA polymerases that can replicate damaged DNA, interacts physically and genetically with the transcription elongation factor NusA (8, 9). ΔdinB strains are sensitive to DNA-damaging agents, nitrofurazone (NFZ) and 4-nitroquinolone-1-oxide (4-NQO), and DinB preferentially and accurately bypasses a structural analog of the major NFZ-induced N 2 -dG lesion as well as certain other N 2 -dG adducts (10-13). NusA is an essential, multidomain protein that functions in both termination and antitermination of transcription, and is associated with the RNA polymerase (RNAP) throughout the elongation and termination phases of transcription (14-22). We have proposed a model of transcription-coupled translesion synthesis (...

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“…In the latter two cases, the gaps would be smaller, 12 to 13 nucleotides (12). DinB has recently been shown to be capable of accurately filling the gaps that could be generated during the repair of N 2 ,N 2 -guanine intrastrand cross-links (24), an observation that might help rationalize the involvement of DNA Pol kappa in mammalian nucleotide excision repair (36).…”

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confidence: 99%

“…E. coli DinB is known to be involved in the phenomenon of untargeted mutagenesis (4) and adaptive mutagenesis (19), and when expressed at increased levels, it causes an increase in Ϫ1 frameshift mutations (22). It was recently discovered that ⌬dinB strains are sensitive to the DNA-damaging agents nitrofurazone and 4-nitroquinolone-1-oxide and that DinB preferentially and accurately bypasses certain N 2 -dG adducts (21,24,32,51). This ability to preferentially bypass these N 2 -dG adducts is conserved evolutionarily (21), suggesting a possible reason for the conservation across all domains of life.…”

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confidence: 99%