Genetic Analysis of Repair and Damage Tolerance Mechanisms for DNA-Protein Cross-Links in Escherichia coli

Abstract

DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually bulky nature. We have recently shown that nucleotide excision repair (NER) and RecBCD-dependent homologous recombination (HR) collaboratively alleviate the lethal effect of DPCs in Escherichia coli. In this study, to gain further insight into the damage-processing mechanism for DPCs, we assessed the sensitivities of a panel of repair-deficient E. coli mutants to DPC-inducing agents, including formaldehyde (FA) and 5-azacytidine (azaC). We show here that the damage tolerance mechanism involving HR and subsequent replication restart (RR) provides the most effective means of cell survival against DPCs. Translesion synthesis does not serve as an alternative damage tolerance mechanism for DPCs in cell survival. Elimination of DPCs from the genome relies primarily on NER, which provides a second and moderately effective means of cell survival against DPCs. Interestingly, Cho rather than UvrC seems to be an effective nuclease for the NER of DPCs. Together with the genes responsible for HR, RR, and NER, the mutation of genes involved in several aspects of DNA repair and transactions, such as recQ, xth nfo, dksA, and topA, rendered cells slightly but significantly sensitive to FA but not azaC, possibly reflecting the complexity of DPCs or cryptic lesions induced by FA. UvrD may have an additional role outside NER, since the uvrD mutation conferred a slight azaC sensitivity on cells. Finally, DNA glycosylases mitigate azaC toxicity, independently of the repair of DPCs, presumably by removing 5-azacytosine or its degradation product from the chromosome.The DNA molecules of living organisms continuously suffer from various types of damage resulting from exposure to endogenous and environmental genotoxic agents. Damage to DNA impairs the faithful propagation of genetic information during replication and transcription, exerting deleterious effects on cells (20). DNA-protein cross-links (DPCs) are unique among DNA lesions in that they are extremely bulky compared to conventional bulky lesions, such as pyrimidine photodimers and the base adducts of aromatic compounds. DPCs are produced by a number of chemical agents, such as aldehydes and heavy metal ions, and also by physical agents such as ionizing radiation and UV light (reviewed in reference 3). DPCs have also been identified in cells or nuclei treated with antitumor agents (4, 10, 44, 62). In addition, we have shown that oxanine, which is produced by nitrosative damage to guanine, mediates the formation of DPCs and polyamine cross-link adducts (49, 50, 52). Thus, understanding the repair and/or damage tolerance mechanism of this ubiquitous and unique class of DNA lesions will provide further insight into how cells maintain genetic integrity and ensure survival in the face of genomic insults. However, the repair and damage tolerance mechanisms of DPCs have long remained elusive, partly because many but not all DPC-inducing agents produce other types of DNA lesions simultaneously, making it rather...

Translocation and Stability of Replicative DNA Helicases upon Encountering DNA-Protein Cross-links

“…The dissociation of stalled DnaB from DNA observed in the present study accounts at least in part for the inactivation of the replisome in vitro, although the in vitro half-lives of stalled replisome (4 -6 min) and DnaB (36 min) differ considerably. The inactivation of the replisome due to loss of DnaB also seems to be consistent with the observations that reactivation of stalled replication fork requires reloading of DnaB (or replication machinery) via the PriA helicase in E. coli (57) and that the priA mutant is hypersensitive to DPC-inducing agents (58). However, further studies are necessary to account for the discrepancy between the in vitro and in vivo stabilities of roadblocked E. coli replisomes (see also below).…”

Modulation of UvrD Helicase Activity by Covalent DNA-Protein Cross-links

“…(concentration of formaldehyde causing 90% cell death) of ϳ5 mM formaldehyde, a result that is consistent with a previous study done in a different strain background (45). These data confirm that UvrD is critical for limiting DPC-induced cytotoxicity.…”

“…Two independent studies (37,45) have also demonstrated that uvrA mutants are sensitive to formaldehyde. One of these studies examined the formaldehyde sensitivity of both ⌬uvrD and ⌬uvrA strains, but these strains were generated from two different parental backgrounds.…”

“…The biochemical pathways by which these lesions are repaired and/or tolerated are not well understood; however, the importance of DNA helicases for cellular tolerance after formaldehyde exposure has recently been demonstrated (24,45).…”

T7 RNA Polymerases Backed up by Covalently Trapped Proteins Catalyze Highly Error Prone Transcription

“…In mammalian cells the upper size limit of crosslinked proteins (CLPs) amenable to NER is around 8 kDa, eliminating the role of NER in the repair of DPCs in vivo (9). DPCs not amenable to NER are processed by homologous recombination in bacterial and mammalian cells (2,7,9,10). These data clearly demonstrate that DPCs impose steric hindrances on DNA polymerases and NER factors, impairing replication and repair.…”