Structural determinants of murine leukemia virus reverse transcriptase that affect the frequency of template switching.
Retroviral reverse transcriptases (RTs) frequently switch templates within the same RNA or between copackaged viral RNAs to generate mutations and recombination. To identify structural elements of murine leukemia virus RT important for template switching, we developed an in vivo assay in which RT template switching within direct repeats functionally reconstituted the green fluorescent protein gene. We quantified the effect of mutations in the YXDD motif, the deoxynucleoside triphosphate binding site, the thumb domain, and the RNase H domain of RT and hydroxyurea treatment on the frequencies of template switching. Hydroxyurea treatment and some mutations in RT increased the frequency of RT template switching up to fivefold, while all of the mutations tested in the RNase H domain decreased the frequency of template switching by twofold. Based on these results, we propose a dynamic copy choice model in which both the rate of DNA polymerization and the rate of RNA degradation influence the frequency of RT template switching.
Svarovskaia
E S
ES
Department of Biochemistry, West Virginia University, Morgantown, West Virginia 26506, USA.
Delviks
K A
KA
Hwang
C K
CK
Pathak
V K
VK
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017353:Gene Deletion; D006918:Hydroxyurea / Q000494:pharmacology; D009052:Leukemia Virus, Murine / Q000201:enzymology* / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D016914:Ribonuclease H / Q000737:chemistry; D013698:Templates, Genetic
D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H; D006918:Hydroxyurea