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Nucleic Acids Research 2005 33(1):171-181; doi:10.1093/nar/gki150
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Published online 12 January 2005

© 2005, the authors Nucleic Acids Research, Vol. 33 No. 1 © Oxford University Press 2005; all rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use permissions, please contact journals.permissions{at}oupjournals.org.

Article

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

Arkadiusz Bibillo, Daniela Lener, George J. Klarmann and Stuart F. J. Le Grice*

Resistance Mechanisms Laboratory, RT Biochemistry Section, HIV Drug Resistance Program, National Cancer Institute at Frederick Frederick, MD 21702, USA

*To whom correspondence should be addressed. Tel: +1 301 846 5256; Fax: +1 301 846 6013; Email: slegrice{at}ncifcrf.gov

Received October 1, 2004. Revised November 22, 2004. Accepted December 7, 2004.

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.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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