Model RNA-directed DNA synthesis by avian myeloblastosis virus DNA polymerase and its associated RNase H

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Abstract
A model RNA template .cntdot. primer system is described for the study of RNA-directed double-stranded DNA synthesis by purified avian myeloblastosis virus DNA polymerase and its associated RNase H. In the presence of complementary RNA primer, oligo(rI), and the deoxyribonucleoside triphosphates dGTP, dTTP and dATP, 3''-(rC)30-40-poly(rA) directs the sequential synthesis of poly(dT) and poly(dA) from a specific site at the 3'' end of the RNA template. With this model RNA template .cntdot. primer, optimal conditions for double-stranded DNA synthesis are described. Analysis of the kinetics of DNA synthesis shows that initially there is rapid synthesis of poly(dT). After a brief time lag, poly(dA) synthesis and the DNA polymerase-associated RNase H activity are initiated. While poly(rA) directs the synthesis of poly(dT), the requirements for DNA synthesis indicate that the newly synthesized poly(dT) acts as template for poly(dA) synthesis. Selective inhibitor studies using NaF show that activation of RNase H is not just a time-related event, but is required for synthesis of the anticomplementary strand of DNA. To determine the specific role of RNase H in this synthetic sequence, the primary for poly(dA) synthesis was investigated. Use of formamide-polyacrylamide slab gel electrophoresis shows that poly(dT) is not acting as both template and primer for poly(dA) synthesis since to poly(dT)-poly(dA) covalent linkages are observed in radioactive poly(dA) product. Identification of 2'',3''-[32P]AMP on paper chromatograms of alkali-treated poly(dA) product synthesized with [.alpha.-32P]dATP as substrate demonstrates the presence of rAMP-dAMP phosphodiester linkages in the poly(dA) product. Therefore, a new functional role of RNase H is demonstrated in the RNA-directed synthesis of double-stranded DNA. Not only in RNase H responsible for the degradation of poly(rA) following formation of a poly(rA) .cntdot. poly(dT) hybrid, but the poly(rA) fragments generated serve as primers for initiation of synthesis of the second strand of the double-stranded DNA.