RESUMEN
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.
Asunto(s)
Archaeoglobus fulgidus/enzimología , Replicación del ADN , Ribonucleasa H/química , Ribonucleasa H/fisiología , Secuencia de Aminoácidos , Ácido Aspártico/química , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Cloruros/farmacología , Secuencia Conservada , Relación Dosis-Respuesta a Droga , Endodesoxirribonucleasas/química , Endonucleasas de ADN Solapado , Iones , Cinética , Cloruro de Magnesio/farmacología , Compuestos de Manganeso/farmacología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Ribonucleasa H/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
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.
