Effects of primer-template sequence on ATP-dependent removal of chain-terminating nucleotide analogues by HIV-1 reverse transcriptase.
HIV-1 reverse transcriptase can remove chain terminators from blocked DNA ends through a nucleotide-dependent mechanism. We show that the catalytic efficiency of the removal reaction can vary several hundred-fold in different sequence contexts and is most strongly affected by the nature of the base pair at the 3'-primer terminus and the six base pairs upstream of it. Similar effects of the upstream sequence were observed with primer-templates terminated with 2',3'-dideoxy-AMP, 2',3'-dideoxy-CMP, or 2',3'-dideoxy-GMP. However, the removal of 2',3'-dideoxy-TMP or 3'-azido-2',3'-dideoxy-TMP was much less influenced by upstream primer-template sequence, and the rate of excision of these thymidylate analogues was greater than or equal to that of the other chain-terminating residues in each sequence context tested. These results strongly indicate that the primer terminus and adjacent upstream base pairs interact with reverse transcriptase in a sequence-dependent manner that affects the removal reaction. We conclude that primer-template sequence context is a major factor to consider when evaluating the removal of different chain terminators by HIV-1 reverse transcriptase.
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33136-1015, USA.
Peter R
PR
Meyer
Anthony J
AJ
Smith
Suzanne E
SE
Matsuura
Walter A
WA
Scott
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism*; D054306:Dideoxynucleotides; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D008969:Molecular Sequence Data; D013698:Templates, Genetic
D017931:DNA Primers; D003838:Deoxyadenine Nucleotides; D054306:Dideoxynucleotides; C010110:2',3'-dideoxyadenosine 5'-phosphate; D000255:Adenosine Triphosphate; D054303:HIV Reverse Transcriptase