DNA nicks inflicted by restriction endonucleases are repaired by a RecA- and RecB-dependent pathway in Escherichia coli.
Two mutants of the EcoRI endonuclease (R200K and E144C) predominantly nick only one strand of the DNA substrate. Temperature sensitivity of the mutant enzymes allowed us to study the consequences of inflicting DNA nicks at EcoRI sites in vivo. Expression of the EcoRI endonuclease mutants in the absence of the EcoRI methyltransferase induces the SOS DNA repair response and greatly reduces viability of recA56, recB21 and lexA3 mutant strains of Escherichia coli. In parallel studies, overexpression of the EcoRV endonuclease in cells also expressing the EcoRV methyltransferase was used to introduce nicks at non-cognate EcoRV sites in the bacterial genome. EcoRV overproduction was lethal in recA56 and recB21 mutant strains and moderately toxic in a lexA3 mutant strain. The toxic effect of EcoRV overproduction could be partially alleviated by introduction into the cells of multiple copies of the E. coli DNA ligase gene. These observations suggest that an increased number of DNA nicks can overwhelm the repair capacity of DNA ligase, resulting in the conversion of a proportion of DNA nicks into DNA lesions that require recombination for repair.
Heitman
J
J
Departments of Genetics, Pharmacology and Cancer Biology, Microbiology, and Medicine and the Howard Hughes Medical Institute, Duke University Medical Center, Box 3546, 322 Carl Building, Durham, NC 27710, USA.
Ivanenko
T
T
Kiss
A
A
D004249:DNA Damage; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics / Q000502:physiology*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D009154:Mutation; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D013014:SOS Response, Genetics; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism; D013696:Temperature
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C056127:DNA modification methylase EcoRV; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D011938:Rec A Recombinases; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; D011088:DNA Ligases