Mechanism of action of Moloney murine leukemia virus RNA-directed DNA polymerase associated RNase H (RNase H I)

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Abstract
The mechanism of action of the RNase H activity associated with Moloney murine leukemia virus RNA-directed DNA polymerase (RNase H-I) and the 2-subunit (.alpha..beta.) form of avian myeloblastosis virus DNA polymerase were compared by utilizing the model substrate (A)n .cntdot. (dT)n and polyacrylamide gel electrophoresis in 7 M urea to analyze digestion products. Examination on 25% polyacrylamide gels revealed that a larger proportion of the RNase H-I oligonucleotide products generated by limited digestion of [3H](A)(1100) .cntdot. (dT)n were acid insoluble (15-26 nucleotides long) than acid soluble (< 5 nucleotides long), while the opposite was true for products generated by .alpha..beta. RNase H. RNase H-I was capable of attacking RNA in RNA .cntdot. DNA in the 5'' to 3'' and 3'' to 5'' directions, as demonstrated by the use of [3H,3''- or 5''-32P](A)(380) .cntdot. (dT)n and cellulose-[3H](A)n .cntdot. (dT)n. Both RNase H I and .alpha..beta. RNase H degraded [3H]-(A-n .cntdot. (dT)n with a partially processive mechanism, based on classical substrate competition experiments and analyses of the kinetics of degradation of [3H,3''- or 5''-32P](A)(380) .cntdot. (dT)n. Both enzymes remain bound to a RNA .cntdot. DNA substrate through a finite number of hydrolytic events but dissociate before the RNA is completely degraded. Both RNase H I and .alpha..beta. RNase H were capable of degrading [14C](A)n in [3H](C)n-[32P](dA)n .cntdot. (dT)n; retroviral RNase H is evidently capable of removing the tRNA primer at the 5'' terminus of minus strand DNA at the appropriate time during retroviral DNA synthesis in vitro.