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Functional roles of carboxylate residues comprising the DNA polymerase active site triad of Ty3 reverse transcriptase.

Abstract：

:Aspartic acid residues comprising the -D-(aa) n -Y-L-D-D- DNA polymerase active site triad of reverse transcriptase from the Saccharomyces cerevisiae long terminal repeat-retrotransposon Ty3 (Asp151, Asp213 and Asp214) were evaluated via site-directed mutagenesis. An Asp151-->Glu substitution showed a dramatic decrease in catalytic efficiency and a severe translocation defect following initiation of DNA synthesis. In contrast, enzymes harboring the equivalent alteration at Asp213 and Asp214 retained DNA polymerase activity. Asp151-->Asn and Asp213-->Asn substitutions eliminated both polymerase activities. However, while Asp214 of the triad could be replaced by either Asn or Glu, introducing Gln seriously affected processivity. Mutants of the carboxylate triad at positions 151 and 213 also failed to catalyze pyrophosphorolysis. Finally, alterations to the DNA polymerase active site affected RNase H activity, suggesting a close spatial relationship between these N- and C-terminal catalytic centers. Taken together, our data reveal a critical role for Asp151 and Asp213 in catalysis. In contrast, the second carboxylate of the Y-L-D-D motif (Asp214) is not essential for catalysis, and possibly fulfills a structural role. Although Asp214 was most insensitive to substitution with respect to activity of the recombinant enzyme, all alterations at this position were lethal for Ty3 transposition.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Bibillo A,Lener D,Klarmann GJ,Le Grice SF

doi

10.1093/nar/gki150

keywords:

subject

Has Abstract

pub_date

2005-01-12 00:00:00

pages

171-81

issue

1

eissn

0305-1048

issn

1362-4962

pii

33/1/171

journal_volume

33

pub_type

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