Cho Endonuclease Functions during DNA Interstrand Cross-Link Repair in Escherichia coli

Abstract

DNA interstrand cross-links are complex lesions that covalently link both strands of the duplex DNA. Lesion removal is proposed to be initiated via the UvrABC nucleotide excision repair complex; however, less is known about the subsequent steps of this complex repair pathway. In this study, we characterized the contribution of nucleotide excision repair mutants to survival in the presence of psoralen-induced damage. Unexpectedly, we observed that the nucleotide excision repair mutants exhibit differential sensitivity to psoralen-induced damage, with uvrC mutants being less sensitive than either uvrA or uvrB. We show that Cho, an alternative endonuclease, acts with UvrAB and is responsible for the reduced hypersensitivity of uvrC mutants. We find that Cho's contribution to survival correlates with the presence of DNA interstrand cross-links, rather than monoadducts, and operates at a step after, or independently from, the initial incision during the global repair of psoralen DNA adducts from the genome. IMPORTANCEDNA interstrand cross-links are complex lesions that covalently bind to both strands of the duplex DNA and whose mechanism of repair remains poorly understood. In this study, we show that Cho, an alternative endonuclease, acts with UvrAB and participates in the repair of DNA interstrand cross-links formed in the presence of photoactivated psoralens. Cho's contribution to survival correlates with the presence of DNA interstrand cross-links and operates at a step after, or independently from, the initial incision during the repair process. P soralens are tricyclic asymmetrical compounds containing furan and pyrone rings and bind DNA nonspecifically, with a preference for pyrimidines to form noncovalent bonds (1-3). Upon absorption of UV-A light, a covalent bond forms through photoaddition between the C-5AC-6 double bond of the pyrimidine and the C-4=AC-5= furan double bond or C-3=AC-4= pyrone double bond of psoralen. Absorption of a second photon results in photoaddition on the remaining furan or pyrone with a second pyrimidine, creating a DNA interstrand cross-link. Thus, psoralen-induced damage consists of both monoadducts and DNA interstrand cross-links. Because DNA interstrand crosslinks covalently bind both strands of the duplex DNA, they inhibit cellular processes that require strand denaturation, including transcription and replication. This inhibition is generally considered to be the reason for the potency of psoralens, and this class of compounds is used in treating different skin diseases, such as vitiligo, psoriasis, and as a chemotherapeutic for some forms of cancer (4-6).Several models have been proposed for DNA interstrand crosslink repair. A feature common to most models is that the repair process is initiated by nucleotide excision repair, followed by the sequential action of other DNA repair processes, such as recombination or translesion synthesis, which function to provide an undamaged template that replaces the incised sequence. In these models, a second round of nucleotide exc...

A bacterial DNA repair pathway specific to a natural antibiotic

“…). It is important to note that B. subtilis uvrABC functioning as a single pathway differs from E. coli (Lage et al ., ; Perera et al ., ).…”

“…In E. coli there is a second UvrC‐like protein called Cho that can also perform the incision function (Moolenaar et al ., ; Perera et al ., ). Mono‐adducts and intrastrand cross‐links are removed from the DNA via UvrD helicase in E. coli after UvrC excision.…”

A bacterial DNA repair pathway specific to a natural antibiotic

“…We found 195 that deletion of mrfA or mrfB rendered B. subtilis hypersensitive to MMC in the absence of 196 uvrAB (Fig 4A), uvrC, or uvrABC ( Fig 4B). We also verified that uvrABC indeed function as a 197 single pathway using an epistasis analysis ( Fig S4), which differs from E. coli (Lage,Goncalves,198 Souza, de Padula, & Leitao, 2010; Perera et al, 2016). To test whether deletion of mrfAB have 199 an effect on acute treatment with MMC, we performed an epistasis analysis using a MMC 200 survival assay.…”

Limited Capacity or Involvement of Excision Repair, Double-Strand Breaks, or Translesion Synthesis for Psoralen Cross-Link Repair in Escherichia coli

“…DNA monoadducts and interstrand cross-links are formed by 8-methoxypsoralen in a ratio of 4:1 (Kanne et al 1982;Tessman et al 1985). Angelicin is a congener of psoralen that, due to its angular structure, is unable to form cross-links and produces almost entirely monoadducts (Dall'Acqua et al 1971;Bordin et al 1976;Ashwood-Smith and Grant 1977;Perera et al 2016). Using this approach, mutants lacking gene products that specifically contribute to the repair of interstrand cross-links would be expected to exhibit a greater sensitivity to 8-methoxypsoralen relative to angelicin when compared to sensitivities of wild-type cultures.…”