Structural biochemistry of a type 2 RNase H: RNA primer recognition and removal during DNA replication

DNA replication and cellular survival requires efficient removal of RNA pri mers during lagging strand DNA synthesis. In eukaryotes, RNA primer removal is initiated by type 2 RNase H, which specifically cleaves the RNA portion of an RNA-DNA/DNA hybrid duplex. This conserved type 2 RNase H family of r eplicative enzymes shares little sequence similarity with the well-characte rized prokaryotic type 1 RNase H enzymes, yet both possess similar enzymati c properties. Crystal structures and structure-based mutational analysis of RNase HII from Archaeoglobus fulgidus, both with and without a bound metal ion, identify the active site for type 2 RNase H enzymes that provides the general nuclease activity necessary for catalysis. The two-domain architec ture of type 2 RNase H creates a positively charged binding groove and link s the unique C-terminal helix-loop-helix cap domain to the active site cata lytic domain. This architectural arrangement apparently couples directional A-form duplex binding, by a hydrogen-bonding Arg-Lys phosphate ruler motif , to substrate-discrimination, by a tyrosine finger motif, thereby providin g substrate-specific catalytic activity. Combined kinetic and mutational an alyses of structurally implicated substrate binding residues validate this binding mode. These structural and mutational results together suggest a mo lecular mechanism for type 2 RNase H enzymes for the specific recognition a nd cleavage of RNA in the RNA-DNA junction within hybrid duplexes, which re conciles the broad substrate binding affinity with the catalytic specificit y observed in biochemical assays. Ln combination with a recent independent structural analysis, these results furthermore identify testable molecular hypotheses for the activity and function of the type 2 RNase H family of en zymes, including structural complementarity, substrate-mediated conformatio nal changes and coordination with subsequent FEN-1 activity. (C) 2001 Acade mic Press.