Effects of single-strand DNases ExoI, RecJ, ExoVII, and SbcCD on homologous recombination of recBCD+ strains of Escherichia coli and roles of SbcB15 and XonA2 ExoI mutant enzymes.
To assess the contributions of single-strand DNases (ssDNases) to recombination in a recBCD+ background, we studied 31 strains with all combinations of null alleles of exonuclease I (delta xon), exonuclease VII (xseA), RecJ DNase (recJ), and SbcCD DNase (sbcCD) and exonuclease I mutant alleles xonA2 and sbcB15. The xse recJ sbcCD delta xon and xse recJ sbcCD sbcB15 quadruple mutants were cold sensitive, while the quadruple mutant with xonA2 was not. UV sensitivity increased with ssDNase deficiencies. Most triple and quadruple mutants were highly sensitive. The absence of ssDNases hardly affected P1 transductional recombinant formation, and conjugational recombinant production was decreased (as much as 94%) in several cases. Strains with sbcB15 were generally like the wild type. We determined that the sbcB15 mutation caused an A183V exchange in exonuclease motif III and identified xonA2 as a stop codon eliminating the terminal 8 amino acids. Purified enzymes had 1.6% (SbcB15) and 0.9% (XonA2) of the specific activity of wild-type Xon (Xon+), respectively, with altered activity profiles. In gel shift assays, SbcB15 associated relatively stably with 3' DNA overhangs, giving protection against Xon+. In addition to their postsynaptic roles in the RecBCD pathway, exonuclease I and RecJ are proposed to have presynaptic roles of DNA end blunting. Blunting may be specifically required during conjugation to make DNAs with overhangs RecBCD targets for initiation of recombination. Evidence is provided that SbcB15 protein, known to activate the RecF pathway in recBC strains, contributes independently of RecF to recombination in recBCD+ cells. DNA end binding by SbcB15 can also explain other specific phenotypes of strains with sbcB15.
Thoms
Brigitte
B
Genetics, Department of Biology and Environmental Sciences, Carl von Ossietzky University Oldenburg, D-26111 Oldenburg, Germany.
Borchers
Inka
I
Wackernagel
Wilfried
W
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003227:Conjugation, Genetic; D003433:Crosses, Genetic; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D043211:Exodeoxyribonuclease V / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005838:Genotype; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D014161:Transduction, Genetic
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C070765:recJ protein, Bacteria; D043211:Exodeoxyribonuclease V