Abstract
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.
Highlights
By using a DNA substrate with defined gap size, we are found in equimolar amounts
Synthesis-To measure strand displacement DNA synthesis, DNA Polymerases-RT activity was determined in a final volume of
The data for the HIV-RT heterodimer p66/p51 are in agreement with a n earlier reportby Huber et al (1989).This activity is not affected by calf thymus PCNA and RF-C or E. coli SSB
Summary
By using a DNA substrate with defined gap size, we are found in equimolar amounts This activity was not af- subunits were separately expressed, they displayed reverse fected first bycalf thymus proliferating cell nuclear ant-ranscriptase activity (Bathurstet al., 1990), but in a hettigenandreplicationfactor C andsecondby Esch- erodimer complex of HIV-RT p66/p51, the HIV-RT p51 subunit erichia coZisingle-strandedDNAmbinding protein, which together allow DNA polymerase 6 to perform stranddisappears to be On the other catalytically silent (El Dirani-Diab et al 1992). F1993) NucZeic Acida Res. 21,841-848).3'-Azido-2',3'dideoxythymidine triphosphate inhibited displacement completely, indicating that DNA synthesis is required forthisreaction.The HIV-RT p66polypeptidealone is able to perform DNA dependent DNA synthesis (Starneset al., 1988) It had been demonstratetdhat e x a ~ natio of the individual subunitisn a variety of templates underconditions optimized for each subunitrevealed a significant catan could perform limited strand displacemDeNntA synthe- lytic activity for the naturalHIV-RT p51 subuni(tThimmig and sis, whereas theHIV-RTpS1 polypeptide was completelyMcHenry, 1993). Kinetic experiments after comple- as s~ng~e-strandepdrimed M13 DNA, but could enhance the mentation ofHIV-RTpsSwith HIV-RTp61 indicated that rate and extentof strand ~ s p ~ a c e m eanctivity of HIV-RT p66, HWRT pS1 can restore rate and extent of strand displacement activity by HN-RT psS compared with the HIV-RT heterodimer p66/pS1, suggesting a function of the 61-kDa polypeptide
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