A distinct form of ribonuclease H from calf thymus stimulates its homologous DNA‐polymerase‐α–primase complex

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Abstract
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‐α–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 α 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.