Requirements for the catalysis of strand transfer synthesis by retroviral DNA polymerases.
We have examined the properties of reverse transcriptases (RTs) required for strand transfer synthesis on poly(rA). In this process, a primer is elongated on one template and then switches to other templates for additional elongation until it is much longer than the templates on which it was made. Models of retrovirus replication require the RT to catalyze two distinct strand transfers. Additionally, they propose that the RT ribonuclease H (RNase H) activity is involved in both transfers. RTs from human immunodeficiency virus (HIV), avian myeloblastosis virus, and murine leukemia virus differ in molecular mass and subunit composition. However, they all catalyzed strand transfer synthesis on (rA)300, generating characteristically long products. An RNase H-deficient enzyme, HIV-RTRD, catalyzed strand transfer synthesis to the same degree as native HIV-RT, indicating that a functional RNase H activity is not required. Additionally, N-ethylmaleimide, which inhibits RNase H but not polymerase activity of HIV-RT, did not diminish strand transfer synthesis. Highly processive DNA synthesis by each RT was found to be required for the strand transfer reaction. RNase H- murine leukemic virus RT has a structural modification that not only eradicates RNase H, but also makes the polymerase much less processive for DNA synthesis. However, conditions that allow this modified enzyme to bind repeatedly to the same primer during synthesis, i.e. conditions that simulate higher processivity, allow strand transfer synthesis. Catalysis of strand transfer synthesis is not a property of all DNA polymerases, since the Klenow fragment of Escherichia coli DNA polymerase I is unable to catalyze this reaction even if high processivity is simulated. These results suggest that strand transfer synthesis relies on an unidentified functional activity present in RTs.
Buiser
R G
RG
Department of Biochemistry, University of Rochester, New York 14642.
DeStefano
J J
JJ
Mallaber
L M
LM
Fay
P J
PJ
Bambara
R A
RA
D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D004256:DNA Polymerase I / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006678:HIV / Q000201:enzymology; D007700:Kinetics; D008979:Moloney murine leukemia virus / Q000201:enzymology; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D012190:Retroviridae / Q000201:enzymology*; D016914:Ribonuclease H
D012194:RNA-Directed DNA Polymerase; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H