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

J Mol Biol. 2001 Mar 23;307(2):541-56. doi: 10.1006/jmbi.2001.4494.


DNA replication and cellular survival requires efficient removal of RNA primers 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 replicative enzymes shares little sequence similarity with the well-characterized prokaryotic type 1 RNase H enzymes, yet both possess similar enzymatic 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 architecture of type 2 RNase H creates a positively charged binding groove and links the unique C-terminal helix-loop-helix cap domain to the active site catalytic 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 providing substrate-specific catalytic activity. Combined kinetic and mutational analyses of structurally implicated substrate binding residues validate this binding mode. These structural and mutational results together suggest a molecular mechanism for type 2 RNase H enzymes for the specific recognition and cleavage of RNA in the RNA-DNA junction within hybrid duplexes, which reconciles the broad substrate binding affinity with the catalytic specificity observed in biochemical assays. In 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 enzymes, including structural complementarity, substrate-mediated conformational changes and coordination with subsequent FEN-1 activity.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Archaeal Proteins / chemistry
  • Archaeal Proteins / metabolism
  • Archaeoglobus fulgidus / enzymology*
  • Catalysis
  • Catalytic Domain
  • Cloning, Molecular
  • Cobalt
  • Crystallography, X-Ray
  • DNA Mutational Analysis
  • DNA Replication*
  • Kinetics
  • Metalloproteins
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Structure, Secondary
  • RNA*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Ribonuclease H / chemistry*
  • Ribonuclease H / genetics
  • Ribonuclease H / metabolism
  • Sequence Homology, Amino Acid


  • Archaeal Proteins
  • Metalloproteins
  • RNA primers
  • Recombinant Proteins
  • Cobalt
  • RNA
  • ribonuclease HII
  • Ribonuclease H

Associated data

  • PDB/1I39
  • PDB/1I3A