Strand displacement activity of the human immunodeficiency virus type 1 reverse transcriptase heterodimer and its individual subunits.
By using a DNA substrate with defined gap size, we found that human immunodeficiency virus type 1 reverse transcriptase (HIV-RT) was able to perform strand displacement DNA synthesis. This activity was not affected first by calf thymus proliferating cell nuclear antigen and replication factor C and second by Escherichia coli single-stranded DNA-binding protein, which together allow DNA polymerase delta to perform strand displacement DNA synthesis (Podust, V., and Hübscher, U. (1993) Nucleic Acids Res. 21, 841-846). 3'-Azido-2',3'-dideoxythymidine triphosphate inhibited displacement completely, indicating that DNA synthesis is required for this reaction. The HIV-RT p66 polypeptide alone could perform limited strand displacement DNA synthesis, whereas the HIV-RT p51 polypeptide was completely inactive, likely due to its inability to replicate extensively on a M13 DNA template. On the other hand the HIV-RT p51 polypeptide enhanced the strand displacement activity of the HIV-RT p66 subunit at a molar ratio of 4:1, mainly by chasing short products into longer ones. Furthermore, kinetic experiments after complementation of HIV-RT p66 with HIV-RT p51 indicated that HIV-RT p51 can restore rate and extent of strand displacement activity by HIV-RT p66 compared with the HIV-RT heterodimer p66/p51, suggesting a function of the 51-kDa polypeptide.
Hottiger
M
M
University Zürich-Irchel, Department of Veterinary Biochemistry, Switzerland.
Podust
V N
VN
Thimmig
R L
RL
McHenry
C
C
Hübscher
U
U
D004247:DNA / Q000378:metabolism; D006678:HIV / Q000201:enzymology*; D054303:HIV Reverse Transcriptase; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013698:Templates, Genetic
D046911:Macromolecular Substances; D004247:DNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase