Nucleic Acids Research Advance Access originally published online on December 12, 2006
Nucleic Acids Research 2007 35(1):256-268; doi:10.1093/nar/gkl909
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Nucleic Acids Research, 2007, Vol. 35, No. 1 256-268
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Purine analog substitution of the HIV-1 polypurine tract primer defines regions controlling initiation of plus-strand DNA synthesis
RT Biochemistry Section, Resistance Mechanisms Laboratory, 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 September 8, 2006. Revised October 11, 2006. Accepted October 12, 2006.
Despite extensive study, the mechanism by which retroviral reverse transciptases (RTs) specifically utilize polypurine tract (PPT) RNA for initiation of plus-strand DNA synthesis remains unclear. Three sequence motifs within or adjacent to the purine-rich elements are highly conserved, namely, a rU:dA tract region immediately 5' to the PPT, an rA:dT-rich sequence constituting the upstream portion of the PPT and a downstream rG:dC tract. Using an in vitro HIV-1 model system, we determined that the former two elements define the 5' terminus of the (+)-strand primer, whereas the rG:dC tract serves as the primary determinant of initiation specificity. Subsequent analysis demonstrated that G
A or AG substitution at PPT positions –2, –4 and +1 (relative to the scissile phosphate) substantially reduces (+)-strand priming. We explored this observation further using PPT substrates substituted with a variety of nucleoside analogs [inosine (I), purine riboside (PR), 2-aminopurine (2-AP), 2,6-diaminopurine (2,6-DAP), isoguanine (iG)], or one of the naturally occurring bases at these positions. Our results demonstrate that for PPT positions –2 or +1, substituting position 2 of the purine was an important determinant of cleavage specificity. In addition, cleavage specificity was greatly affected by substituting –4G with an analog containing a 6-NH2 moiety.
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