Motif IV and V of Active DNA Dependent ATPase a Domain, a SWI2/SNF2 Protein, Are Required for Both Ligand Binding and Conformational Integrity

Abstract

ATP dependent chromatin remodeling proteins use the energy released by ATP hydrolysis to alter the chromatin structure to make it accessible for fundamental processes e.g. replication, transcription and repair etc. SMARCAL1 is a distant member of SNF2 family of chromatin remodelers and has been reported to interact with RPA at the DNA damage site and exhibits reannealing helicase activity and maintain the genome integrity. Mutations in hSMARCAL cause a rare autosomal recessive disorder known as Schimke Immunoosseous Dysplasia (SIOD). ADAAD is the N-terminal proteolytic fragment of SMARCAL1 that possesses all the characteristic helicase motifs (Q, I, Ia, II, III, IV, V and VI) of SMARCAL. It can hydrolyse the ATP only in presence of a stem-loop DNA structure. Using ADAAD I created F507A and F507W site directed mutant of motif IV and S558A and T560A site directed mutant of motif V of ADAAD to investigate the function of these motifs in the ADAAD. Results showed that F507A was inactive whereas S558A and T560A had the same activity as compare to ADAAD. Kinetic studies of S558A and T560A showed that the kinetic parameters of S558A and T560A were lowered as compared to ADAAD. So these residues are important for the turnover number and catalytic effieciency of the ADAAD. Binding studies showed that F507A binding was lowered with either ligand in presence of another ligand. Circular dichroism studies showed that the conformation of these mutants were altered as compare to ADAAD. Thermal denaturation data by circular dichroism showed that Tm was almost same of all these mutants as compare to ADAAD. In conclusion, motif IV and V are required for the conformational integrity of the protein.