A distinct form of ribonuclease H from calf thymus stimulates its homologous DNA-polymerase-alpha-primase complex

Eur J Biochem. 1989 Nov 20;185(3):621-8. doi: 10.1111/j.1432-1033.1989.tb15158.x.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cattle
  • Centrifugation, Density Gradient
  • DNA / biosynthesis
  • DNA Primase
  • DNA Replication
  • DNA-Directed DNA Polymerase / isolation & purification*
  • Electrophoresis, Gel, Two-Dimensional
  • Endoribonucleases / isolation & purification*
  • Endoribonucleases / pharmacology
  • Enzyme Activation / drug effects
  • Hydrolysis
  • Magnesium
  • RNA Nucleotidyltransferases / isolation & purification*
  • Ribonuclease H
  • Substrate Specificity
  • Thymus Gland / enzymology*
  • Thymus Gland / metabolism


  • DNA
  • DNA Primase
  • RNA Nucleotidyltransferases
  • DNA-Directed DNA Polymerase
  • Endoribonucleases
  • Ribonuclease H
  • Magnesium