Translesion DNA polymerases are required for spontaneous deletion formation in Salmonella typhimurium.
How spontaneous deletions form in bacteria is still a partly unresolved problem. Here, we show that deletion formation in Salmonella typhimurium requires the presence of functional translesion polymerases. First, in wild-type bacteria, removal of the known translesion DNA polymerases, PolII (polB), PolIV (dinB), PolV (umuDC), and SamAB (samAB), resulted in a 10-fold decrease in the deletion rate, indicating that 90% of all spontaneous deletions require these polymerases for their formation. Second, overexpression of these polymerases by derepression of the DNA damage-inducible LexA regulon caused a 25-fold increase in deletion rate that depended on the presence of functional translesion polymerases. Third, overexpression of the polymerases PolII and PolIV from a plasmid increased the deletion rate 12- to 30-fold, respectively. Last, in a recBC(-) mutant where dsDNA ends are stabilized due to the lack of the end-processing nuclease RecBC, the deletion rate was increased 20-fold. This increase depended on the translesion polymerases. In lexA(def) mutant cells with constitutive SOS expression, a 10-fold increase in DNA breaks was observed. Inactivation of all 4 translesion polymerases in the lexA(def) mutant reduced the deletion rate 250-fold without any concomitant reduction in the amount of DNA breaks. Mutational inactivation of 3 endonucleases under LexA control reduced the number of DNA breaks to the wild-type level in a lexA(def) mutant with a concomitant 50-fold reduction in deletion rate. These findings suggest that the translesion polymerases are not involved in forming the DNA breaks, but that they require them to stimulate deletion formation.
Koskiniemi
Sanna
S
Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
Andersson
Dan I
DI
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D053960:DNA Breaks; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D018085:Regulon; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics*; D017384:Sequence Deletion; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004259:DNA-Directed DNA Polymerase; D011938:Rec A Recombinases; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein