HIGH-AFFINITY INTERACTION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE WITH PARTIALLY COMPLEMENTARY PRIMERS

The comparison of K-m and V-max values for various primers in the reac tion of polymerization catalyzed by the human immunodeficiency virus t ype-1 (HIV-1) reverse transcriptase was carried out. The primers were: (a) complementary to the template, (b) partially complementary with m ismatched nucleotides at different positions from the 3' end or (c) no n-complementary. Non-complementary primers were not elongated by HIV-1 reverse transcriptase. However, if they contained only one residue co mplementary to the template or an abasic unit at the 3' end, they coul d serve as primers. The most effective discrimination between marched and mismatched primers, due to a decrease in the affinity and V-max, w as found in the case of oligonucleotides containing non-complementary bases at the second or third position from the 3' end of the primer. T he 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 archaebacteri al DNA polymerases and avian myeloblastosis virus reverse transcriptas e. Oligonucleotides such as (dT)(4)(dCdG)(k)(dT)(4)) showed higher aff inity for the enzyme than (dT)(4) or (dT)(8) primers. These data sugge st that HIV-1 reverse transcriptase, in contrast to procaryotic, eucar yotic and archaebacterial DNA polymerases, forms additional contacts w ith the 5'-end region of the non-complementary primer. In addition, us ing tRNA(3)(Lys), the natural primer of HIV-1, it was shown that the p 66 subunit of reverse transcriptase can be crosslinked, in the presenc e of a platinum derivative, to the 5' end of tRNA. Thus, besides the n ormal binding site for the 3' end of tRNA, which is crucial for the in itiation of cDNA synthesis, the 5' end of the tRNA also interacts with a specific site on the enzyme.