Archaeoglobus fulgidus RNase HII in DNA Replication: Enzymological Functions and Activity Regulation via Metal Cofactors
| Autor: | Qing Chai, Junzhuan Qiu, Binghui Shen, Brian R. Chapados |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
DNA Replication
Ribonucleotide Flap Endonucleases RNase P Molecular Sequence Data Ribonuclease H Magnesium Chloride Biophysics Binding Competitive Biochemistry Substrate Specificity Chlorides Amino Acid Sequence Binding site RNase H Molecular Biology Conserved Sequence Ions Aspartic Acid Nuclease Binding Sites Endodeoxyribonucleases Base Sequence Dose-Response Relationship Drug Sequence Homology Amino Acid biology Archaeoglobus fulgidus DNA replication Cell Biology Kinetics RNase MRP Manganese Compounds Mutation Mutagenesis Site-Directed biology.protein Protein Binding |
| Zdroj: | Biochemical and Biophysical Research Communications. 286:1073-1081 |
| ISSN: | 0006-291X |
| DOI: | 10.1006/bbrc.2001.5523 |
| Popis: | 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 Mg2+ and Mn2+. 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. |
| Databáze: | OpenAIRE |
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