Nucleic Acids Research, 2003, Vol. 31, No. 5 1481-1487
© 2003 Oxford University Press
The ribonuclease H activity of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2 is modulated by residue 294 of the small subunit
Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel and 1 Department of Chemistry, Institute of Catalysis, Science and Technology, Technion, Israel Institute of Technology, Haifa 32000, Israel
*To whom correspondence should be addressed. Tel: +972 3 6409974; Fax: +972 3 6407432; Email: ahizy{at}post.tau.ac.il
Reverse transcriptases (RTs) exhibit DNA polymerase and ribonuclease H (RNase H) activities. The RTs of human immunodeficiency viruses type 1 and type 2 (HIV-1 and HIV-2) are composed of two subunits, both sharing the same N-terminus (which encompasses the DNA polymerase domain). The smaller subunit lacks the C-terminal segment of the larger one, which contains the RNase H domain. The DNA polymerase domain of RTs resembles a right hand linked to the RNase H domain by a connection subdomain. Despite the high homology between HIV-1 and HIV-2 RTs, the RNase H activity of the latter is substantially lower than that of HIV-1 RT. The thumb subdomain of the small subunit controls the level of RNase H activity. We show here that Gln294, located in this thumb, is responsible for this difference in activity. A HIV-2 RT mutant, where Gln294 in the small subunit was replaced by a proline (present in HIV-1 RT), has an activity almost 10-fold higher than that of the wild-type RT. A comparative in vitro study of the kinetic parameters of the RNase H activity suggests that residue 294 affects the Km rather than the kcat value, influencing the affinity for the RNA·DNA substrate.
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