Different Mode of Action of Ribonuclease H in Purified α and αβ Ribonucleic Acid-directed Deoxyribonucleic Acid Polymerase from Avian Myeloblastosis Virus

Abstract The mechanism of action of ribonuclease H (RNase H) associated with the two structurally different RNA-directed DNA polymerases of avian myeloblastosis virus was studied. Both the two-subunit enzyme, αβ, and the single subunit enzyme, α, require free termini to initiate nuclease activity since they cannot cleave a covalently closed RNA-DNA duplex. The exoribonuclease activity of both α and αβ RNase H can degrade RNA of an RNA-DNA hybrid in the 3' → 5' and 5' → 3' directions, as shown by the use of 3H, 3'- or 5'-32Ppoly(A)·poly(dT) as substrate. Two oligonucleotide species approximately 10 and 30 nucleotides long are generated by limited digestion of 3Hpoly(A)·poly(dT) by both enzymes. But α and αβ possess a different mode of exoribonuclease activity. αβ RNA-directed DNA polymerase, as shown previously, has processive exoribonuclease activity which degrades one polyribonucleotide chain completely prior to initiating hydrolysis of a second chain. In contrast, α RNA-directed DNA polymerase has random exoribonuclease activity which results in the release of the substrate molecule after each chain scission. This conclusion is supported by two types of experiments: (a) while the degradation of radioactive substrate by αβ is unaffected by addition of unlabeled substrate after the reaction has been initiated, the degradation of the labeled polymer by α is immediately blocked, and (b) αβ degrades 3' and 5' end terminally labeled substrate at the same rate as the whole molecule, while α degrades the end terminal nucleotides at a much faster rate than the whole molecule. The β subunit of αβ may facilitate tighter binding of the enzyme to the substrate resulting in a processive mode of nuclease activity.