Functions that protect Escherichia coli from DNA-protein crosslinks.
Pathways for tolerating and repairing DNA-protein crosslinks (DPCs) are poorly defined. We used transposon mutagenesis and candidate gene approaches to identify DPC-hypersensitive Escherichia coli mutants. DPCs were induced by azacytidine (aza-C) treatment in cells overexpressing cytosine methyltransferase; hypersensitivity was verified to depend on methyltransferase expression. We isolated hypersensitive mutants that were uncovered in previous studies (recA, recBC, recG, and uvrD), hypersensitive mutants that apparently activate phage Mu Gam expression, and novel hypersensitive mutants in genes involved in DNA metabolism, cell division, and tRNA modification (dinG, ftsK, xerD, dnaJ, hflC, miaA, mnmE, mnmG, and ssrA). Inactivation of SbcCD, which can cleave DNA at protein-DNA complexes, did not cause hypersensitivity. We previously showed that tmRNA pathway defects cause aza-C hypersensitivity, implying that DPCs block coupled transcription/translation complexes. Here, we show that mutants in tRNA modification functions miaA, mnmE and mnmG cause defects in aza-C-induced tmRNA tagging, explaining their hypersensitivity. In order for tmRNA to access a stalled ribosome, the mRNA must be cleaved or released from RNA polymerase. Mutational inactivation of functions involved in mRNA processing and RNA polymerase elongation/release (RNase II, RNaseD, RNase PH, RNase LS, Rep, HepA, GreA, GreB) did not cause aza-C hypersensitivity; the mechanism of tmRNA access remains unclear.
Department of Biochemistry, Duke University Medical Center, Durham NC 27710, United States.
Rachel
R
Krasich
Department of Biochemistry, Duke University Medical Center, Durham NC 27710, United States.
Sunny Yang
SY
Wu
Department of Biochemistry, Duke University Medical Center, Durham NC 27710, United States.
H Kenny
HK
Kuo
Department of Biochemistry, Duke University Medical Center, Durham NC 27710, United States. Electronic address: kenneth.kreuzer@duke.edu.
Kenneth N
KN
Kreuzer
D001374:Azacitidine / Q000633:toxicity*; D004249:DNA Damage; D004260:DNA Repair; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics; D012329:RNA, Bacterial / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; C096222:tmRNA; D001374:Azacitidine
Azacytidine; Cytosine methyltransferase; DNA helicases; DNA repair; tmRNA