Although there was little to no expression
Although there was little to no expression change observed by TELI of the recA and ruvA genes upon CAA exposure, other specific repair pathways were induced by both CAA and SO and include the SOS-inducible gene sulA, the DNA repair gene ykfG, the mismatch repair gene mutS, the recombination and repair genes recE and recN, the DNA gyrase inhibitor sbmC, and the SOS-induced putative antitoxin symE. The recE gene encodes exonuclease VIII and digests DNA in the 5′ →3′ direction, yielding dsDNA with 3′- ssDNA overhangs and recE mutations cause recB, recC, and sbcA mutants to become recombination-deficient . By TELI, the recE promoter showed increased expression upon exposure to both CAA and SO. We further find by TELI that the sbcB promoter shows increased expression upon exposure to both agents; sbcB encodes exonuclease I, which is involved in the RecBCD pathway . We find that the recN promoter showed increased TELI response, but the strain harboring a recN deletion was not sensitive to either CAA or SO. Taken together, this work implicates recombination repair in cellular responses to CAA and SO.
The promoters of the glycosylases mutM, mug, and the nuclease nfo each showed increased TELI responses upon exposure to CAA and SO and the strains lacking mutM and nfo showed sensitivity to SO. Increased expression of the mug, mutM, and nfo promotors as determined by TELI and the lowered survival of the mutM and nfo mutants suggests the involvement of BER in repair of SO-induced lesions, consistent with the finding that SO-induced lesions lead to formation of abasic sites . Finally, deletion of uvrA and uvrD, both involved in NER, resulted in sensitivity to SO, presumably due to the presence of bulky styrene adducts and consistent with a previous report that lack of uvrA confers modest sensitivity to SO . Like recA, genes involved in NER also confer survival upon exposure to a wide range of agents including radiation and several DBU synthesis [, , , , ].
In summary, we confirmed that a broader range of DNA damage genes were expressed upon exposure to SO, with a number of the same genes expressed upon exposure to CAA by the TELI assay. The survival assays also yielded a broader range of DNA damage response genes that confer resistance to SO, which could be due to the greater diversity of DNA damage induced by SO. The goal of this work was to identify genes important for responses to the two alkylating agents CAA and SO and to point out aspects of DNA damage responses that need further study. For example, the lack of correlation between TELI gene expression and cellular sensitivity for dinG is unexpected, and deletion of ybfE, a gene of unknown function, confers sensitivity to both CAA and SO. In the long term, the technology might be applied to exposure of cell cultures from individuals to learn which chemicals are most toxic on an individual basis.
Conflict of interest statement
Acknowledgments Susan Cohen and Graham Walker of MIT are acknowledged for the gift of several strains used in this work. Bilyana Koleva is acknowledged for technical assistance. Rhodes Technologies is acknowledged for their generous support of M.M.M. This research was supported by the American Cancer Society (RSG-12-161-01-DMC to P.J.B.), ROUTES Fellowships (NIEHS 1R25ES025496) to A.A. and J.O.A., the Northeastern University Office of the Provost for Undergraduate Research and Creative Endeavors Awards to C.J. and S.B. and an Honors Assistantship to K.C.W. This study was also supported by National Science Foundation (NSF, CBET-1437257, CBET-1810769 to A.Z.G.), National Institute of Environmental Health Sciences (NIEHS, PROTECT P42ES017198 and CRECE P50ES026049 to R.W.G. and A.Z.G.), and Environmental Protection Agency (EPA, CRECE 83615501 to A.Z.G.).
Introduction Microorganisms play a vital role in the remediation and detoxification of the environment. They are able to metabolize chemical contaminants that can be completely degraded to end products such as CO2 and H2O, or transformed to compounds with lower solubility and toxicity. Exposure to chemicals and radiation introduces lesions to DNA that can be cytotoxic, mutagenic or neutral to the cell. Some lesions block DNA polymerases, leading to replication fork arrest and cell death, whereas others may miscode during replication causing mutations in newly synthesized DNA. To counteract the cytotoxic and mutagenic effects of chemicals, cells have evolved a number of mechanisms for the repair and maintenance of DNA integrity which involve many proteins dedicated to this task. Alkylating agents damage DNA by direct action. Present in the environment as endogenous by-products of cellular metabolism (including lipid peroxidation), they are also used as chemotherapeutics.