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Journal of Virology, July 2001, p. 6337-6347, Vol. 75, No. 14
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.14.6337-6347.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

A Ty1 Reverse Transcriptase Active-Site Aspartate Mutation Blocks Transposition but Not Polymerization†

Ozcan Uzun1 and Abram Gabriel2,*

Graduate Program in Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey,1 and Department of Molecular Biology and Biochemistry, Rutgers University,2 Piscataway, New Jersey 08854

Received 28 December 2000/Accepted 12 April 2001

Reverse transcriptases (RTs) are found in a wide variety of mobile genetic elements including viruses, retrotransposons, and infectious organellar introns. An invariant triad of aspartates is thought to be required for the catalytic function of RTs. We generated RT mutants in the yeast retrotransposon Ty1, changing each of these active-site aspartates to asparagine or glutamate. All but one of the mutants lacked detectable polymerase activity. The novel exception, D211N, retained near wild-type in vitro polymerase activity within virus-like particles but failed to carry out in vivo transposition. For this mutant, minus-strand synthesis is impaired and formation of the plus-strand strong-stop intermediate is eliminated. Intragenic second-site suppressor mutations of the transposition defect map to the RNase H domain of the enzyme. Our results demonstrate that one of the three active-site aspartates in a retrotransposon RT is not catalytically critical. This implies a basic difference in the polymerase active-site geometry of Ty1 and human immunodeficiency virus RT and shows that subtle mutations in one domain can cause dramatic functional effects on a distant domain of the same enzyme.

* Corresponding author. Mailing address: Department of Molecular Biology and Biochemistry, Rutgers University, CABM 306, 679 Hoes Lane, Piscataway, NJ 08854. Phone: (732) 235-5097. Fax: (732) 235-4880. E-mail: gabriel{at}cabm.rutgers.edu.

dagger Dedicated to the memory of Esther M. Gabriel.

Journal of Virology, July 2001, p. 6337-6347, Vol. 75, No. 14
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.14.6337-6347.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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