Analysis of mutations made during active synthesis or extension of mismatched substrates further define the mechanism of HIV-RT mutagenesis.
The effect of reverse transcriptase (RT) catalyzed mutations on continued extension of the nascent DNA chain was investigated. A system using the alpha-lac gene of beta-galactosidase as template and two sets of conditions was used. In one, RT was allowed to reassociate with the primer-template after falling off, while in a second RT was sequestered after dissociating. In the first condition, subsequent extension of errors that may have initially caused enzyme dissociation can occur. In the second, such errors would not be extended. Fully extended products were assayed by alpha-complementation to assess mutation frequency. A lower frequency in the latter scenario implies that some errors caused the polymerase to dissociate. Allowing only a single binding event lowered the mutation frequency of the products by about 1/2 suggesting that approximately 1 in 2 errors terminated synthesis. In other experiments, when added to a primer-template with a terminal mismatch at the 3' end, RT dissociated from the template about 50-90% of the time (depending on mismatch type) rather than extending. Running start reactions indicated that extension was more likely if an actively synthesizing RT made the mutation. RT RNase H cleavage analysis showed that 3' mismatches weakened the association of RT with the primer-terminus. Taken together, these results suggest that an actively synthesizing RT enzyme that has just made a mistake is likely bound in a configuration that generally enhances extension of the mistake. This is in contrast to RTs that must bind to then extend mismatches. The importance of these findings with respect to the mechanism of mutagenesis is discussed.
Liesch
Gwyneth R
GR
Department of Cell Biology and Molecular Genetics (CBMG), University of Maryland-College Park, College Park, Maryland 20742, USA.
DeStefano
Jeffrey J
JJ
D020137:Base Pair Mismatch; D001483:Base Sequence; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000235:genetics* / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D016296:Mutagenesis; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D013379:Substrate Specificity
D004279:DNA, Viral; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H