Site-Directed Mutagenesis법에 의한 RecA 단백질의 ATPase 활성 부위 검정 : Cysteinyl 129의 관련성 Involvement of Cysteinyl 129 = Determination of ATPase Active Site for RecA Protein by Site-directed Mutagenesis

다국어 초록 (Multilingual Abstract)

ATPase activity of RecA protein is believed to be essential for the strand exchange and DNA repair reactions in E. coli. Chemical modification(Kuramitsu et al., Biochemistry. 23. 2363. 1984) and ATP photolabeling(Banks and Sedgwick. Biochemistry, 25, 5882, 1986) experiments suggest that cysteinyl-129 is one of the most probable residues for the binding and hydrolysis of ATP in RecA protein. Site-directed mutagenesis is employed to change cysteinyl-129 to serine and alanine to minimize any structural alterations. Wild type and mutant RecA proteins were purified and examined for ATPase activity. All the purified RecA proteins showed sigmoidal activity shapes as a function of substrate concentration, indicating that RecA catalyzed ATP hydrolysis is cooperative. Both mutant RecAs displayed almost same V_(max) with wild type RecA but much larger S_(1/2) values(l80 μM for Ser. 120 μM for ,A1a) than wild type(78 μM). These results suggest that cysteinyl-129 may involve in the ATP binding rather than ATP hydrolysis. Further experiments on direct ATP binding and strand exchange reactions are in progress.