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  • The TELI results of DNA

    2020-09-18

    The TELI results of DNA damage responses to CAA and SO then informed our choice of bacterial strains in subsequent experiments. We investigated E. coli cellular survival in response to CAA and SO exposure by determining the sensitivity of a number of E. coli strains, each possessing single or multiple gene deletions. We find that multiple repair processes confer resistance to SO while only a few processes confer resistance to CAA.
    Materials and methods
    Results In order to determine the cellular pathways responsible for tolerance to CAA and SO, we determined the expression of stress response genes and assessed the survival of a number of E. coli strains containing deletions of genes that were likely to confer survival. The strains chosen for this work contained deletions of genes that represented a range of DNA repair functions as well as some of the genes implicated in cellular responses to CAA and SO from TELI experiments.
    Discussion In this work, we used assays of gene expression and cellular sensitivity to identify repair pathways that play pivotal roles in tolerance to DNA damage induced by the alkylating agents CAA and SO. There are several types of DNA damage caused by the alkylating agents studied here. For example, DNA exposure to CAA has been shown to result in formation of the mutagenic exocyclic adducts εC, εA, εG, and 1,N2-εG [[8], [9], [10], [11],19,77]. E. coli AlkB repairs εC, εA, and 1,N2-εG adducts, similar to the repair profile of the human homolog ABH2 [9,[18], [19], [20], [21],78,79]. The εC and 1,N2-εG lesions are also repaired by base excision repair proteins double-stranded uracil-DNA glycosylase (UDG) and Mug, respectively; 1,N2-εG does not appear to be a substrate for Nfo or AlkB [9,15]. Some DNA glycosylase genes, such as ung and mutM, exhibit altered expression upon cellular exposure to CAA (Fig. 2). We observed only modest sensitivity to CAA of a strain lacking alkB, which could be due to the presence of compensating repair pathways. Indeed, the most sensitive strains to CAA here were those lacking recombination repair genes recA and ruvA. SO forms adducts via either its epoxide α- or β-position at multiple sites of dA, dC, and dG, and at the N3 position of dT [[5], [6], [7],22,23]. The dG adduct can undergo subsequent depurination, resulting in an abasic site [80], consistent with the finding that endonuclease III is involved in repair of SO-induced damage in human cells [81], and our observation that several BER-associated genes showed TELI responses upon exposure to SO (Fig. 2). In E. coli, elevated SO concentrations promoted acetic Fostamatinib formation, membrane permeability, and cell lysis, and a reduction in colony growth and formation, which together are likely the reasons for the more robust stress response to SO in the TELI experiment [82]. It was observed in in vivo bacterial replication assays that DNA containing most SO lesions could be replicated, but DNA containing some of these same lesions could not be replicated with purified replicative DNA polymerases [83,84]. This previous work was carried out prior to the discovery of the biochemical activity of Y-family translesion DNA polymerases; indeed, we observed that deletion of both Y-family polymerases umuDC and dinB sensitizes cells to SO (Fig. 5), suggesting that they play a role in bypass of SO-induced DNA lesions. Deletions of the genes recA, involved in recombination and repair, and ybfE, a gene of unknown function, conferred sensitivity to both CAA and SO. Although recA promoter activity was essentially unchanged upon CAA exposure, there was a low but detectable expression change upon exposure to SO. A similar observation was made for the ruvA promoter, which showed slightly higher expression when exposed to SO than to CAA. One possible reason for the lack of recA promoter activity in TELI is that recA is one of the most abundant proteins in the cell and thus it has been proposed that sufficient RecA is present for some of its functions without induction [85]. Similarly, of two related carcinogens, N-hydroxy-N-2-aminofluorene and acetoxy-N-2-acetylaminofluorene, only the latter induced RecA [86]. Using TELI, similar effects were observed with two genotoxic nanomaterials, nano-silver and nano-TiO2_a, in which nano-silver did not induce RecA whereas nano-TiO-2_a led to robust induction [[33], [34], [35]]. Although the promoters of recA and ruvA are not appreciably activated by exposure to either agent, the roles of recA and ruvA in recombination and DNA repair [60,65,66,87] appear to be important for survival upon exposure to these agents. The strains lacking recA and ybfE showed the largest degree of sensitivity upon exposure to both agents. The critical and multifaceted roles of recA in stress responses are also highlighted by its contributions to survival upon UV- and X-irradation, as well as exposure to a number of antibiotics and other damaging agents [[88], [89], [90], [91], [92], [93]]. Although the function of ybfE is unknown, the decrease in cell survival observed for the mutant strain when exposed to either agent suggests that it plays a key role in DNA damage tolerance; indeed, the ybfE gene is known to be regulated by LexA [74,94] and was upregulated by both CAA and SO.