A partially deficient mutant, recA1730, that fails to form normal nucleoprotein filaments.
The phenotype of the recA1730 mutant is highly dependent on the level of expression of the RecA1730 protein. If the recA1730 gene was expressed from its own promoter, the cells were deficient in recombination and SOS induction. In contrast, when the recA1730 gene was expressed under the control of recAo98, a constitutive operator that increased the RecA1730 concentration 20-fold, cells became proficient in recombination and SOS induction. Likewise, in crude extracts, fivefold more RecA1730 than RecAwt was required to produce full cleavage of LexA protein. The requirement for a high RecA1730 concentration for recombination and LexA cleavage suggests that the recA1730 defect alters a common reaction step. In fact, in vitro data show that the impaired assembly of RecA1730 protein on single-stranded DNA (ssDNA) can account for the mutant phenotype. Purified RecA1730 protein was assayed in vitro for ssDNA binding and ATPase activities. RecA1730, like RecAwt, retained ssDNA equally well on nitrocellulose filters; this activity was specifically inhibited by a monoclonal anti-RecA antibody. However, RecA1730 protein did not form complete filaments on ssDNA, as shown by two observations: (i) most of the protein did not elute with ssDNA during gel filtration; and (ii) binding of RecA1730 to ssDNA did not protect it from being digested by DNaseI. RecA1730 hydrolysed ATP in high salt but was defective in ssDNA-dependent ATP hydrolysis. These results strongly suggest that RecA1730 binds to ATP and ssDNA but does not form normal nucleoprotein filaments.
Dutreix
M
M
Groupe d'Etude Mutagénèse et Cancerogénèse, Laboratoire d'Enzymologie, CNRS, Gif-sur-Yvette, France.
Burnett
B
B
Bailone
A
A
Radding
C M
CM
Devoret
R
R
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000483:Alleles; D001426:Bacterial Proteins / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D005798:Genes, Bacterial; D007700:Kinetics; D008970:Molecular Weight; D016296:Mutagenesis; D009698:Nucleoproteins / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011938:Rec A Recombinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D012697:Serine Endopeptidases; D014466:Ultraviolet Rays; D000099160:LexA Repressor Protein
D000251:Adenosine Triphosphatases; D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D009698:Nucleoproteins; D011938:Rec A Recombinases; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein