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Biochem Biophys Res Commun. 2001 Sep 7;286(5):1073-81.

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

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1
Department of Cell and Tumor Biology, City of Hope National Medical Center, Duarte, California 91010, USA.

Abstract

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.

PMID:
11527410
DOI:
10.1006/bbrc.2001.5523
[Indexed for MEDLINE]

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