Archaeoglobus fulgidus RNase HII in DNA replication: enzymological functions and activity regulation via metal cofactors

Biochem Biophys Res Commun. 2001 Sep 7;286(5):1073-81. doi: 10.1006/bbrc.2001.5523.


RNA primer removal during DNA replication is dependent on ribonucleotide- and structure-specific RNase H and FEN-1 nuclease activities. A specific RNase H involved in this reaction has long been sought. RNase HII is the only open reading frame in Archaeoglobus fulgidus genome, while multiple RNases H exist in eukaryotic cells. Data presented here show that RNase HII from A. fulgidus (aRNase HII) specifically recognizes RNA-DNA junctions and generates products suited for the FEN-1 nuclease, indicating its role in DNA replication. Biochemical characterization of aRNase HII activity in the presence of various divalent metal ions reveals a broad metal tolerance with a preference for Mg(2+) and Mn(2+). Combined mutagenesis, biochemical competitions, and metal-dependent activity assays further clarify the functions of the identified amino acid residues in substrate binding or catalysis, respectively. These experiments also reveal that Asp129 form a second-metal binding site, and thus contribute to activity attenuation.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Archaeoglobus fulgidus / enzymology*
  • Aspartic Acid / chemistry
  • Base Sequence
  • Binding Sites
  • Binding, Competitive
  • Chlorides / pharmacology
  • Conserved Sequence
  • DNA Replication*
  • Dose-Response Relationship, Drug
  • Endodeoxyribonucleases / chemistry
  • Flap Endonucleases
  • Ions
  • Kinetics
  • Magnesium Chloride / pharmacology
  • Manganese Compounds / pharmacology
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation
  • Protein Binding
  • Ribonuclease H / chemistry*
  • Ribonuclease H / metabolism
  • Ribonuclease H / physiology*
  • Sequence Homology, Amino Acid
  • Substrate Specificity


  • Chlorides
  • Ions
  • Manganese Compounds
  • Magnesium Chloride
  • Aspartic Acid
  • Endodeoxyribonucleases
  • Flap Endonucleases
  • ribonuclease HII
  • Ribonuclease H
  • manganese chloride