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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 

Incorporation of uracil into minus strand DNA affects the specificity of plus strand synthesis initiation during lentiviral reverse transcription.

Many retroviruses either encode dUTP pyrophosphatase (dUTPase) or package host-derived uracil DNA glycosylase as a means to limit the accumulation of uracil in DNA strands, suggesting that uracil is detrimental to one or more steps in the viral life cycle. In the present study, the effects of DNA uracilation on (-) strand DNA synthesis, RNase H activity, and (+) strand DNA synthesis were investigated in a cell-free system. This system uses the activities of purified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase to convert single-stranded RNA to double-stranded DNA in a single reaction mixture. Substitution of dUTP for dTTP had no effect on (-) strand synthesis but significantly decreased yields of (+) strand DNA. Mapping of nascent (+) strand 5' ends revealed that this was due to decreased initiation from polypurine tracts with a concomitant increase in initiation at non-polypurine tract sites. Aberrant initiation correlated with a change in RNase H cleavage specificity when assayed on preformed RNA-DNA duplexes containing uracilated DNA, suggesting that appropriate "selection" of the (+) strand primer is affected. Collectively, these data suggest that accumulation of uracil in retroviral DNA may disrupt the viral life cycle by altering the specificity of (+) strand DNA synthesis initiation during reverse transcription.[1]

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