Chromosomal fragmentation in dUTPase-deficient mutants of Escherichia coli and its recombinational repair.
Recent findings suggest that DNA nicks stimulate homologous recombination by being converted into double-strand breaks, which are mended by RecA-catalysed recombinational repair and are lethal if not repaired. Hyper-rec mutants, in which DNA nicks become detectable, are synthetic-lethal with recA inactivation, substantiating the idea. Escherichia coli dut mutants are the only known hyper-recs in which presumed nicks in DNA do not cause inviability with recA, suggesting that nicks stimulate homologous recombination directly. Here, we show that dut recA mutants are synthetic-lethal; specifically, dut mutants depend on the RecBC-RuvABC recombinational repair pathway that mends double-strand DNA breaks. Although induced for SOS, dut mutants are not rescued by full SOS induction if RecA is not available, suggesting that recombinational rather than regulatory functions of RecA are needed for their viability. We also detected chromosomal fragmentation in dut rec mutants, indicating double-strand DNA breaks. Both the synthetic lethality and chromosomal fragmentation of dut rec mutants are suppressed by preventing uracil excision via inactivation of uracil DNA-glycosylase or by preventing dUTP production via inactivation of dCTP deaminase. We suggest that nicks become substrates for recombinational repair after being converted into double-strand DNA breaks.
Department of Microbiology, University of Illinois at Urbana-Champaign, B103 C & LSL, 601 South Goodwin Ave., Urbana, IL 61801-3709, USA.
Elena A
EA
Kouzminova
Andrei
A
Kuzminov
D000595:Amino Acid Sequence; D002876:Chromosomes, Bacterial; D004249:DNA Damage; D053938:DNA Fragmentation; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D011755:Pyrophosphatases / Q000235:genetics* / Q000378:metabolism; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D013014:SOS Response, Genetics; D016415:Sequence Alignment; D014498:Uracil / Q000378:metabolism
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D014498:Uracil; D011938:Rec A Recombinases; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase