Abstract
Abstract A ribonuclease degrading only RNA complexed to DNA is found associated with the avian myeloblastosis virus DNA polymerase. A convenient and sensitive assay for the enzyme is degradation of [3H]poly(A) complexed to poly(dT). Using this assay, nuclease and DNA polymerase activities are inseparable by DEAE-Sephadex or phosphocellulose ion exchange chromatography or by glycerol gradient centrifugation. Poly(A) labeled selectively at each end can be used to demonstrate that the nuclease is an endonuclease, and chromatography of the digestion products of poly(A) confirms this result. The oligonucleotide digestion products can be further digested to 5'-AMP by venom phosphodiesterase, indicating that they are terminated by 3'-OH groups.
Highlights
Poly(A) labeled selectively at each end can be used to demonstrate that the nuclease is an endonuclease, and chromatography of the digestion products of poly(A) confirms this result
Many bacterial DNA polymerases have associated m-ith them nuclease activity (l-5) the major mammalian DNA polymerases appear to lack such an activity [6].’. This activity is exonucleolytic and it has been suggested that the liuclease serves an editing function, excising improper nucleotides in order that the polymerizing activity can replace them with the proper nucleotides [4, 7]
Synthesis-dependent Degradation of Poly(B)-Ha\ing previously analyzed some of t’hr propertics of poly(A) as a template for poly(dT) synthesis by the AMV DNA polymerase (11, la), WC decided to investigate the stability of the template during synthesis
Summary
A ribonuclease degrading only RNA complexed to DNA is found associated with the avian myeloblastosis virus DNA polymerase. Using this assay, nuclease and DNA polymerase activities are inseparable by DEAE-Sephadex or phosphocellulose ion exchange chromatography or by glycerol gradient centrifugation. Polymerase Assay-The standard assay for polymerase activity [11] was 0.1 ml containing 50 mM Tris-HCl (pH 8.3), 6 MM magnesium acetate, 20 rnitf dithiothreitol, 60 mM NaCl, 3 nmoles of poly(A), 1.5 nmoles of poly(dT)jz-18, and 18 nmoles of [3H]dTTP (61 cpm per pmole). To determine the number of units of polymerase activity, a reaction system of 0.1 ml was used with the same buffer and salts as above but containing 2.6 nmoles of poly(C), 1.35 nmoles of poly(dG)12-is, and 20 nmoles of [3H]dGTI’ (65 cpm per pmole). Samples were prepared for counting as described [22]
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