A distinct form of ribonuclease H from calf thymus stimulates its homologous DNA-polymerase-alpha-primase complex.
A ribonuclease H which degrades RNA specifically in RNA-DNA hybrids and, moreover, stimulates its homologous DNA-polymerase-primase complex was purified from calf thymus. The enzyme consists of a single polypeptide of molecular mass 78 kDa. It requires divalent cations for activity, and prefers Mg2+ over Mn2+. Ribonuclease H is optimally active at neutral pH and in 75 mM potassium acetate and is strongly sensitive to N-ethylmaleimide. [3H]Poly(rA).poly(dT), [3H]poly(rC).poly(dI), and [3H]RNA.M13-DNA are degraded to 3-9-mer oligoribonucleotides with similar kinetics, whereas double- or single-stranded DNA, and double- and single-stranded RNA remain unaffected. The enzyme stimulates in vitro DNA synthesis by the immunoaffinity-purified calf-thymus DNA-polymerase-alpha-primase complex threefold. When ribonuclease H is present in a three-fold molar excess to the polymerase-primase complex, twice as much primer is formed as in the absence of ribonuclease H. Ribonuclease H also stimulates the elongation rate of DNA polymerase alpha by a factor of 2-3, independent of whether primase-primed DNA templates or templates primed with oligonucleotides are used. Our results suggest that this form of ribonuclease H is a likely candidate for a genuine primer-removing enzyme in mammalian cells.
Hagemeier
A
A
Department of Chemistry, Max-Planck-Institute for Experimental Medicine, Göttingen, FRG.
Grosse
F
F
D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000096:biosynthesis; D019915:DNA Primase; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional; D004722:Endoribonucleases / Q000302:isolation & purification* / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006868:Hydrolysis; D008274:Magnesium; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification*; D016914:Ribonuclease H; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology* / Q000378:metabolism
D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004259:DNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H; D008274:Magnesium