New mutations in and around the L2 disordered loop of the RecA protein modulate recombination and/or coprotease activity

Abstract

The RecA protein plays a key role in Escherichia coli recombination and DNA repair. We have created new recA mutants with mutations in the vicinity of the recA430 mutation which is known to affect RecA coprotease activity. Mutants carrying recA659 or recA611, located 3 and 7 amino acids downstream of residue 204, respectively, lose all RecA activities, while the mutant carrying recA616, which is located at 12 amino acids from this residue, keeps the coprotease activity but is unable to promote recombination. Complementation experiments show that both mutations recA611 and recA659 are dominant over the wild-type or recA430 allele while recA616 seems to be recessive to recA+ and dominant over recA430. It is suggested that these mutations are located in RecA domains which direct conformational modifications.

CTXφ contains a hybrid genome derived from tandemly integrated elements

“…Consequently, we assessed whether homologous recombination is necessary for production of CTX virions by strains with tandem prophages, using two recA Ϫ derivatives of BD81. One derivative, BD206, has a 2.8-kb deletion encompassing most of recA whereas the other, BD578, has a point mutation (K215Q) expected to impair homologous recombination but not RecA-mediated protease activity (16). We found that supernatants from BD206 yielded significantly fewer transductants than the isogenic wild type strain, although Kn R colonies were still obtained with an average frequency of 420 transductants ϫ OD 600 Ϫ1 ϫ ml Ϫ1 (Fig.…”

Investigation of the Secondary DNA-binding Site of the Bacterial Recombinase RecA

“…Upon AgeI digestion, the amplified DNA fragment was cloned into the large fragment of AgeIdigested pCCrecA to give pCCE207K recombinant plasmid. recA E207Q (14,20) was cloned in the same vector.…”

“…SOS induction was measured using a bacterial strain carrying a cea::lacZ fusion and measuring ␤-galactosidase expression as described elsewhere (14,15). The induction factor is the ratio of ␤-galactosidase activities after and before irradiation.…”

“…The induction factor is the ratio of ␤-galactosidase activities after and before irradiation. Intrachromosomal and interchromosomal recombination assays were measured using published protocols (14).…”

“…To address this question, we have investigated two mutant RecA proteins, RecA E207Q (14) and RecA E207K (this work), that eliminate the negative charge at position 207 and replace it with an uncharged or positively charge residue, respectively. We have compared the capacities of these proteins for DNA repair, SOS induction, and homologous recombination in vivo as well as the DNA strand exchange and DNA-dependent ATPase activities of purified proteins in vitro.…”