Mutating conserved residues in the ribonuclease H domain of Ty3 reverse transcriptase affects specialized cleavage events.
The reverse transcriptase-associated ribonuclease H (RT/RNase H) domains from the gypsy group of retrotransposons, of which Ty3 is a member, share considerable sequence homology with their retroviral counterparts. However, the gypsy elements have a conserved tyrosine (position 459 in Ty3 RT) instead of the conserved histidine in the catalytic center of retroviral RTs such as at position 539 of HIV-1. In addition, the gypsy group shows conservation of histidine adjacent to the third of the metal-chelating carboxylate residues, which is Asp-426 of Ty3 RT. The role of these and additional catalytic residues was assessed with purified recombinant enzymes and through the ability of Ty3 mutants to support transposition in Saccaromyces cerevisiae. Although all mutations had minimal impact on DNA polymerase function, amidation of Asp-358, Glu-401, and Asp-426 eliminated Mg(2+)- and Mn(2+)-dependent RNase H function. Replacing His-427 and Tyr-459 with Ala and Asp-469 with Asn resulted in reduced RNase H activity in the presence of Mg(2+), whereas in the presence of Mn(2+) these mutants displayed a lack of turnover. Despite this, mutations at all positions were lethal for transposition. To reconcile these apparently contradictory findings, the efficiency of specialized RNase H-mediated events was examined for each enzyme. Mutants retaining RNase H activity on a heteropolymeric RNA.DNA hybrid failed to support DNA strand transfer and release of the (+) strand polypurine tract primer from (+) RNA, suggesting that interrupting one or both of these events might account for the transposition defect.
Lener
Daniela
D
Reverse Transcriptase Biochemistry Section, Resistance Mechanisms Laboratory, HIV Drug Resistance Program, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702, USA.
Budihas
Scott R
SR
Le Grice
Stuart F J
SF
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002478:Cells, Cultured; D017124:Conserved Sequence; D004926:Escherichia coli; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D018626:Retroelements; D016914:Ribonuclease H / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D013329:Structure-Activity Relationship
D018626:Retroelements; D008345:Manganese; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H; D008274:Magnesium