title: Disruption of an ATP-dependent isomerization of the recA protein by mutation of histidine 163.
abstract: We have used site-directed mutagenesis to replace histidine 163 of the recA polypeptide with an alanine residue. The new [Ala-163]recA protein catalyzes single-stranded (ss) DNA-dependent ATP hydrolysis with a turnover number that is similar to that of the wild-type recA protein. Despite being proficient in ssDNA-dependent ATP hydrolysis, the [Ala-163]recA protein is unable to promote the ATP-dependent three-strand exchange reaction under standard reaction conditions, pH 7.5. The [Ala-163]recA protein does exhibit three-strand exchange activity at pH 6.0-7.0, however, and the induction of strand exchange activity at low pH correlates directly with the activation of an ATP-dependent isomerization of the mutant protein. Thus, the [Ala-163]recA protein is functionally similar to our previously described mutant [Asn-160]recA protein (Bryant, F.R. (1988) J. Biol. Chem. 263, 8716-8723; Muench, K.A., and Bryant, F. R. (1990) J. Biol. Chem. 265, 11560-11566). Trypsin proteolysis studies indicate that the [Ala-163]recA and [Asn-160]recA proteins, like the wild-type recA protein, are organized into carboxyl-terminal and amino-terminal domains of nearly equal size. According to this structural model, the [Ala-163]recA and [Asn-160]recA mutations may lie in a linker region joining these two domains. We speculate that the [Ala-163]recA and [Asn-160]recA mutations interfere with an ATP-dependent conformational change of the recA protein that perhaps involves a change in the relative orientation of the carboxyl-terminal and amino-terminal domains.
authors.affiliation: Department of Biochemistry, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205.
authors.forename: K A
authors.initials: KA
authors.lastname: Muench
authors.forename: F R
authors.initials: FR
authors.lastname: Bryant
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000409:Alanine / Q000235:genetics; D004269:DNA, Bacterial / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007536:Isomerism; D009154:Mutation; D011938:Rec A Recombinases / Q000737:chemistry* / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D006639:Histidine; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D000251:Adenosine Triphosphatases; D000409:Alanine