Reengineering the nucleotide cofactor specificity of the RecA protein by mutation of aspartic acid 100

We have recently obtained evidence for a direct linkage between the S_0.5 (S_0.5 is the substrate concentration required for half-maximal velocity) value of a nucleoside triphosphate and the conformational state of the RecA-ssDNA complex, with an S_0.5 value of 125 μM or less required for stabilization of the strand exchange-active conformation. For example, although ATP and ITP are hydrolyzed by the RecA protein with the same turnover number (18 min^-1), ATP (S_0.5 = 45 μM) functions as a cofactor for the strand exchange reaction, whereas ITP (S_0.5 = 500 μM) is inactive as a strand exchange cofactor. The RecA protein crystal structure suggests that cofactor specificity is determined by Asp¹⁰⁰, which likely forms a hydrogen bond with the exocyclic 6-amino group of ATP; the higher S_0.5 value for ITP is presumably due to unfavorable interactions between Asp¹⁰⁰ and the 6-carbonyl group of the inosine ring. To test this hypothesis, we prepared a mutant RecA protein in which Asp¹⁰⁰ was replaced by an asparagine residue. The S_0.5(ITP) for the [D100N]RecA protein is 125 μM, indicating favorable interactions between the Asn¹⁰⁰ side chain and the 6- carbonyl group of ITP. Correspondingly, ITP functions as a cofactor for the strand exchange activity of the [D100N]RecA protein. This result demonstrates the importance of the residue at position 100 in determining nucleotide cofactor specificity and underscores the importance of the S_0.5 value in the RecA protein-promoted strand exchange reaction.