Characterization of ribonuclease H activity associated yeast RNA polymerase a

Huet, J.; Buhler, J.M.; Sentenac, A.; Fromageot, P.

Journal of Biological Chemistry 252(24): 8848-8855

1977


ISSN/ISBN: 0021-9258
PMID: 21884
Accession: 068518255

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Abstract
The ribonuclease H activity associated with yeast RNA polymerase A degrades a variety of RNA-DNA hybrids with apparently no base specificity. The nuclease activity was characterized using (rA)n .cntdot. (dT)n and (rG)n .cntdot. (dC)n hybrids. It shows an absolute requirement for a divalent cation, Mg2+ or Mn2+. The pH curve is bimodal, with optima at pH 6.5 and 8. The optimal temperature depends strongly on the nature of the divalent cation. The activity is inhibited by low salt concentrations, EDTA, N-ethylmaleimide and (r.ovrhdot.I)n. The nuclease activity is also drastically reduced under conditions in which polymerization can proceed, even with limiting concentrations of ribonucleoside triphosphates. RNA polymerase A executes an exonucleolytic attack on the hybridized RNA, producing a mixture of a mononucleotide 5'-phosphate and of a dinucleotide with a 5'-phosphate end. The pattern of degradation products varies strongly with the incubation conditions. Depending on pH and divalent cation used, the dinucleotide/mononucleotide ratio can vary by a factor of 20 or more. During the course of these experiments it was found that Mn2+ ions, in absence of enzyme, catalyze the hydrolysis of (rA)n to a mixture of acid-soluble oligonucleotides of decreasing length, terminated with a 3'-phosphate group. After mild dissociation of RNA polymerase A with urea, the RNase H activity was recovered associated with the fractions containing both dissociated polypeptides, A48 and A34.5 subunits, and with the RNA polymerase A*, which is lacking these subunits. The RNase H activity again comigrated with A* enzyme upon polyacrylamide gel electrophoresis. Enzyme A* produced a mixture of dinucleotides and mononucleotides like Enzyme A, although in a significantly different ratio; and this ratio varied with the incubation conditions. The possibility of 2 distinct RNase H associated with the polymerase is discussed. The role of such ribonuclease H activity is still unclear, however, its presence in yeast RNA polymerase A supports the hypothesis that eukaryotic RNA polymerases are part of a multienzyme system involved in chromatin activity.