Abstract
In this study we examine for the first time the roles of the various domains of human RNase H1 by site-directed mutagenesis. The carboxyl terminus of human RNase H1 is highly conserved with Escherichia coli RNase H1 and contains the amino acid residues of the putative catalytic site and basic substrate-binding domain of the E. coli RNase enzyme. The amino terminus of human RNase H1 contains a structure consistent with a double-strand RNA (dsRNA) binding motif that is separated from the conserved E. coli RNase H1 region by a 62-amino acid sequence. These studies showed that although the conserved amino acid residues of the putative catalytic site and basic substrate-binding domain are required for RNase H activity, deletion of either the catalytic site or the basic substrate-binding domain did not ablate binding to the heteroduplex substrate. Deletion of the region between the dsRNA-binding domain and the conserved E. coli RNase H1 domain resulted in a significant loss in the RNase H activity. Furthermore, the binding affinity of this deletion mutant for the heteroduplex substrate was approximately 2-fold tighter than the wild-type enzyme suggesting that this central 62-amino acid region does not contribute to the binding affinity of the enzyme for the substrate. The dsRNA-binding domain was not required for RNase H activity, as the dsRNA-deletion mutants exhibited catalytic rates approximately 2-fold faster than the rate observed for wild-type enzyme. Comparison of the dissociation constant of human RNase H1 and the dsRNA-deletion mutant for the heteroduplex substrate indicates that the deletion of this region resulted in a 5-fold loss in binding affinity. Finally, comparison of the cleavage patterns exhibited by the mutant proteins with the cleavage pattern for the wild-type enzyme indicates that the dsRNA-binding domain is responsible for the observed strong positional preference for cleavage exhibited by human RNase H1.
Highlights
Two classes of RNase H enzymes have been identified in mammalian cells [5, 9, 10]
In this study we examine for the first time the roles of the various domains of human RNase H1 by site-directed mutagenesis
In this study we have explored the roles of the conserved amino acids of the catalytic site and the basic substrate-binding domain, the roles of the double-strand RNA (dsRNA)-binding domain, and the 62-amino acid center region of human RNase H1 (Fig. 1)
Summary
Two classes of RNase H enzymes have been identified in mammalian cells [5, 9, 10]. These enzymes were shown to differ with respect to cofactor requirements and were shown to be inhibited by sulfhydryl reagents [10, 11]. The biological roles of the mammalian enzymes are not fully understood, it has been suggested that mammalian RNase H1 may be involved in replication and that the RNase H2 enzyme may be involved in transcription [12, 13] Both human RNase H genes have been cloned and expressed [11, 14, 15]. The amino acid sequence of human RNase H1 displays strong homology with RNase H1 from yeast, chicken, Escherichia coli, and mouse [11]. The human RNase H2 enzyme is a 299-amino acid protein with a calculated mass of 33.4 kDa and has been shown to be ubiquitously expressed in human cells and tissues [14].1. The carboxyl-terminal portion of human RNase H1 is highly conserved with the amino acid sequence of the E. coli enzyme (Fig. 1, Region III). The lysine residues within the highly basic ␣-helical substrate-binding region of E. coli RNase H1 are conserved in the human enzyme
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call