Investigating the structure of human RNase H1 by site-directed mutagenesis

In this study we examine for the first time the roles of the various domain s of human RNase H1 by site-directed mutagenesis. The carboxyl terminus of human RNase H1 is highly conserved with Escherichia coli RNase H1 and conta ins the amino acid residues of the putative catalytic site and basic substr ate binding do. main of the E. coli RNase enzyme. The amino terminus of hum an RNase H1 contains a structure consistent with a double-strand RNA (dsRNA ) binding motif that is separated from the conserved E. coli RNase H1 regio n by a Ga-amino acid sequence. These studies showed that although the conse rved amino acid residues of the putative catalytic site and basic substrate -binding domain are required for RNase H activity, deletion of either the c atalytic site or the basic substrate-binding domain did not ablate binding to the heteroduplex substrate. Deletion of the region between the dsRNA-bin ding domain and the conserved E. coli RNase H1 domain resulted in a signifi cant loss in the RNase H activity. Furthermore, the binding affinity of thi s deletion mutant for the heteroduplex substrate was similar to2-fold tight er than the wildtype enzyme suggesting that this central ga-amino acid regi on does not contribute to the binding affinity of the enzyme for the substr ate. The dsRNA-binding domain was not required for RNase H activity, as the dsRNA-deletion mutants exhibited catalytic rates similar to2-fold faster t han the rate observed for wild-type enzyme. Comparison of the dissociation constant of human RNase H1 and the dsRNA-deletion mutant for the heterodupl ex substrate indicates that the deletion of this region resulted in a 5-fol d loss in binding affinity. Finally, comparison of the cleavage patterns ex hibited by the mutant proteins with the cleavage pattern for the wild-type enzyme indicates that the dsRNA-binding domain is responsible for the obser ved strong positional preference for cleavage exhibited by human RNase H1.