An obligatory pH-mediated isomerization on the [Asn-160]recA protein-promoted DNA strand exchange reaction pathway.
We recently described a mutant recA protein in which glycine 160 of the recA polypeptide was replaced by an asparagine residue (Bryant, F. R. (1988) J. Biol. Chem. 263, 8716-8723). Although the [Asn-160]recA protein has a ssDNA-dependent ATPase activity that is similar to that of the wild-type recA protein, the mutant protein is unable to promote the ATP-dependent three-strand exchange reaction under standard reaction conditions (pH 7.5, 1 mM ATP). We have found that the [Asn-160]recA protein is able to carry out the three-strand exchange reaction at pH 6.0 to 6.7, but that the strand exchange activity is abolished at higher pH. The induction of strand exchange activity at low pH correlates directly with a pH-mediated activation of an ATP-dependent isomerization of the [Asn-160]recA protein. This ATP-dependent isomerization is characterized by the conversion of the [Asn-160]recA protein to a form that is not displaced from ssDNA by the Escherichia coli SSB protein. In contrast to the pronounced pH sensitivity of the [Asn-160]recA protein, the wild-type recA protein undergoes ATP-dependent isomerization, and is able to carry out the three-strand exchange reaction, over the range of pH 6.0 to 8.4. These results show that the [Asn-160] mutation disrupts the ATP-dependent isomerization of the recA protein and suggest that protonation of the [Asn-160]recA protein (or the [Asn-160]recA-ssDNA complex) relieves this mechanistic defect. Furthermore, the direct correlation between ATP-dependent isomerization and the strand exchange activity of the [Asn-160]recA protein strongly suggests that the ATP-dependent isomerization is an obligatory step in the recA protein-promoted strand exchange mechanism.
Muench
K A
KA
Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21205.
Bryant
F R
FR
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001216:Asparagine; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007536:Isomerism; D007700:Kinetics; D009154:Mutation; D011938:Rec A Recombinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001216:Asparagine; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D000251:Adenosine Triphosphatases; C037693:single stranded DNA dependent ATPase