Archaeoglobus fulgidus RNase HII in DNA replication: enzymological functions and activity regulation via metal cofactors.
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.
Chai
Q
Q
Department of Cell and Tumor Biology, City of Hope National Medical Center, Duarte, California 91010, USA.
Qiu
J
J
Chapados
B R
BR
Shen
B
B
D000595:Amino Acid Sequence; D019611:Archaeoglobus fulgidus / Q000201:enzymology*; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002712:Chlorides / Q000494:pharmacology; D017124:Conserved Sequence; D004261:DNA Replication; D004305:Dose-Response Relationship, Drug; D004706:Endodeoxyribonucleases / Q000737:chemistry; D045585:Flap Endonucleases; D007477:Ions; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D017895:Manganese Compounds / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D016914:Ribonuclease H / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002712:Chlorides; D007477:Ions; D017895:Manganese Compounds; D015636:Magnesium Chloride; D001224:Aspartic Acid; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; C091139:ribonuclease HII; D016914:Ribonuclease H; C025340:manganese chloride