Y586F mutation in murine leukemia virus reverse transcriptase decreases fidelity of DNA synthesis in regions associated with adenine–thymine tracts

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The National Academy of Sciences

RESUMO

Using in vivo fidelity assays in which bacterial β-galactosidase or green fluorescent protein genes served as reporters of mutations, we have identified a murine leukemia virus (MLV) RNase H mutant (Y586F) that exhibited an increase in the retroviral mutation rate ≈5-fold in a single replication cycle. DNA-sequencing analysis indicated that the Y586F mutation increased the frequency of substitution mutations 17-fold within 18 nt of adenine–thymine tracts (AAAA, TTTT, or AATT), which are known to induce DNA bending. Sequence alignments indicate that MLV Y586 is equivalent to HIV-1 Y501, a component of the recently described RNase H primer grip domain, which contacts and positions the DNA primer strand near the RNase H active site. The results suggest that wild-type reverse transcriptase (RT) facilitates a specific conformation of the template–primer duplex at the polymerase active site that is important for accuracy of DNA synthesis; when an adenine–thymine tract is within 18 nt of the polymerase active site, the Y586F mutant RT cannot facilitate this specific template–primer conformation, leading to an increase in the frequency of substitution mutations. These findings indicate that the RNase H primer grip can affect the template–primer conformation at the polymerase active site and that the MLV Y586 residue and template–primer conformation are important determinants of RT fidelity.

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