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

Abstract

A ribonuclease H which degrades RNA specifically in K N A-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 MgZ+ 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]KNA . MI 3-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 immunoaffinitypurified calf-thymus DNA-polymerase-a -primase complex threefold. When ribonuclease H is present in a threefold molar excess to the polymerase-primase complex, twice as much primer is formed as in the absence of ribonuclease H. Kibonuclease H also stimulates the elongation rate of DNA polymerase L Y by a factor of 2-3. independent of whether primase-primed DNA templates or templates primed with oligonucleotides are used. Our rcsults suggest that this form of ribonuclease H is a likely candidate for a genuine primer-removing enzyme in maininalian cells.DNA replication is a complex process which requires at least 20 -30 different proteins. Central activities for lagging strand synthesis involve a DNA primase for the formation o f short pieces of RNA, typically 5 -10 nucleotides long. which serve as primers for the replicative DNA polymerase (for a review see 11, 21). In eukaryotic organisnis RNA primer formation and DNA synthesis are performed by a multisubunit complex, named DNA polymerase-or -primase complex (for a review see [3]). The removal of RNA primers is a prerequisite for the subsequent sealing of Okazaki fragments by DNA ligation. Most likely, a ribonuclease specific for the RNA strand of a DNA-RNA hybrid, ribonuclease H (RNase H; EC 3.1.4.34), is responsible for primer removal. Such an enzyme was first detected by Skin and Hausen in calf thymus 141. Since then, the puritication and characterization of several different forms of RNase H from many organisms has been reported (for a review see [S]). However, until now it was not clear which of the known forms of RNase H were involved in the process of DNA replication.Our laboratory is concerned with the identification and isolation of proteins involved in DNA replication in calf thymus cells in order to reconstitute a replication complex in vitro. These studies began with the isolation of the DNA polymerase cc [6, 71 and were continued by the isolation and Corresponderrce to F. Grosse, Abteilung Chemie.

Junction ribonuclease: An activity in Okazaki fragment processing

“…Two classes of RNase H, RNase HI and II, have been identified in a variety of eukaryotes, purified and characterized (1)(2)(3)(4)(5)(6)(7)(8) as a cofactor. Both forms of RNase H randomly degrade RNA in long RNA͞DNA hybrid structures (2,9).…”

Single‐stranded DNA binding protein from calf thymus

“…In our recent work we have purified and characterized many replicative proteins from calf thymus glands, including the DNA-polymerase-a -primase complex [13,141, the DNA polymerases 6 and F [I 51, a polymerase-a-stimulating ribonuclease H [16], DNA topoisomerases [17], and two DNA helicases [I 81. Because of the species-specific interaction between SSB and polymerase a and probably other components of the replicative apparatus as well, we attempted to purify SSB from bovine tissue.…”

Ribonuclease H attack of leukaemic fused transcripts AML1‐MTG8(ETO) by DNA/RNA chimeric hammerhead ribozymes

“…The accessibility of anti-sense ONs to intracellular mRNA, however, will be different from that to isolated RNA in vitro, because mRNAs interact with many protein factors and form secondary and tertiary structures in vivo. Heidenreich et al (1995) reported the enhancement of the ribozyme activity in isolated nuclei and thought that it was due to protein factors that protect and help ribozymes annealing to target RNA, such as hnRNP A1 and HIV-1 NC7 protein. Although we only surveyed a 40-ntd region of AML1-MTG8 mRNA adjacent to the fusion point, this region was effectively digested with endogenous RNase H by the addition of anti-sense ONs in nuclei.…”

“…Therefore, RNase HII might be responsible for the anti-sense effect. In addition to these two classes of RNase H enzymes, another distinct form of RNase H has been puri®ed from calf thymus as a stimulating factor of DNA polymerase a-primase activity (Hagemeier & Grosse 1989). This enzyme consists of a single polypeptide of 78 kDa, requiring divalent cations for activity (preferring Mg 2 to Mn 2 ), and is strongly sensitive to NEM.…”

“…HEL cells (a human erythroleukaemia cell line) were grown in RPMI1640 supplemented with 10% foetal calf serum. The nuclei of SKNO-1 and HEL cells were prepared according to Heidenreich et al (1995) with some modi®cations. Approximately 10 8 cells were harvested and washed with phosphate buffered saline and suspended in 0.5 mL of hypotonic buffer (10 mM Tris-HCI, pH 7.5, 5 mM MgCl 2 , 0.3% NP-40, 0.5 mM DTT, 0.5 mM PMSF).…”