Human immunodeficiency virus type-1 reverse transcriptase copies very short templates: kinetic and crosslinking analysis.
We describe in this article some properties concerning the cDNA elongation activity of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT). The kinetic parameters of the polymerization reaction catalyzed by HIV-1 RT, using short templates, were studied. Values of Km and Vmax were measured as a function of the oligoadenylate template length: the logarithm of Km increased linearly, with an incremental factor of 2.2, when the template length differs by one nucleotide. Using short templates, olig(A)n (n = 7-14) and primers shorter or longer than the template, HIV-1 reverse transcriptase was able to synthesize polymer products longer than 200 nucleotides. We showed that an oligonucleotide as short as (pA)3 was long enough to serve as template for cDNA synthesis by RT. In the binding of RT to template of different lengths (5 to 14 nucleotides long), two constants were determined differing in each case by a factor of about 10. The three recombinant forms of HIV-1 RT (p66/p51, p66/p66 and p51/p51) were crosslinked to a short template, (pA)14, in the presence of cis-aquahydroxydiamminoplatinum. The efficiency of crosslink of [32P](pA)14 template with each of the subunits of RT correlated well with the affinity of this template to the different forms of RT. In the case of p66/p51, the crosslink occurred mainly with the p66 subunit. These results confirm the important catalytic role of the p66 subunit in the heterodimeric human retroviral polymerase.
Andreola
M L
ML
Institut de Biochimie Cellulaire du CNRS, Bordeaux, France.
Dufour
E
E
Tarrago-Litvak
L
L
Jamkovoy
V I
VI
Levina
A S
AS
Barr
P J
PJ
Litvak
S
S
Nevinsky
G A
GA
D002945:Cisplatin / Q000031:analogs & derivatives; D003432:Cross-Linking Reagents; D054303:HIV Reverse Transcriptase; D007700:Kinetics; D011061:Poly A; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors; D013379:Substrate Specificity; D013698:Templates, Genetic
D003432:Cross-Linking Reagents; D018894:Reverse Transcriptase Inhibitors; D011061:Poly A; C081461:aquahydroxydiamminoplatinum; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D002945:Cisplatin