High-affinity interaction of human immunodeficiency virus type-1 reverse transcriptase with partially complementary primers.
The comparison of Km and Vmax values for various primers in the reaction of polymerization catalyzed by the human immunodeficiency virus type-1 (HIV-1) reverse transcriptase was carried out. The primers were: (a) complementary to the template, (b) partially complementary with mismatched nucleotides at different positions from the 3' end or (c) non-complementary. Non-complementary primers were not elongated by HIV-1 reverse transcriptase. However, if they contained only one residue complementary to the template or an abasic unit at the 3' end, they could serve as primers. The most effective discrimination between matched and mismatched primers, due to a decrease in the affinity and Vmax, was found in the case of oligonucleotides containing non-complementary bases at the second or third position from the 3' end of the primer. The efficiency of discrimination by HIV-1 reverse transcriptase between matched and mismatched base-paired primers was about 1-1.5 orders of magnitude lower than that of procaryotic, eucaryotic and archaebacterial DNA polymerases and avian myeloblastosis virus reverse transcriptase. Oligonucleotides such as (dT)4(dCdG)k(dT)4 showed higher affinity for the enzyme than (dT)4 or (dT)8 primers. These data suggest that HIV-1 reverse transcriptase, in contrast to procaryotic, eucaryotic and archaebacterial DNA polymerases, forms additional contacts with the 5'-end region of the non-complementary primer. In addition, using tRNA(3Lys), the natural primer of HIV-1, it was shown that the p66 subunit of reverse transcriptase can be crosslinked, in the presence of a platinum derivative, to the 5' end of tRNA. Thus, besides the normal binding site for the 3' end of tRNA, which is crucial for the initiation of cDNA synthesis, the 5' end of the tRNA also interacts with a specific site on the enzyme.
Zakharova
O D
OD
Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, Russia.
Tarrago-Litvak
L
L
Maksakova
G
G
Andréola
M L
ML
Dufour
E
E
Litvak
S
S
Nevinsky
G A
GA
D018076:DNA, Complementary; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D009841:Oligonucleotides; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase