Y586F mutation in murine leukemia virus reverse transcriptase decreases fidelity of DNA synthesis in regions associated with adenine-thymine tracts.
Using in vivo fidelity assays in which bacterial beta-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 approximately 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.
HIV Drug Resistance Program, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA.
Wen-Hui
WH
Zhang
Evguenia S
ES
Svarovskaia
Rebekah
R
Barr
Vinay K
VK
Pathak
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D004261:DNA Replication / Q000502:physiology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007700:Kinetics; D009052:Leukemia Virus, Murine / Q000201:enzymology / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics*; D016914:Ribonuclease H / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H