Mispair extension fidelity of human immunodeficiency virus type 1 reverse transcriptases with amino acid substitutions affecting Tyr115

Abstract

The role of Tyr115 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) in the mispair extension fidelity of DNA dependent DNA synthesis was analysed by using a series of 15 mutant enzymes with substitutions at Tyr115. Their kinetic parameters for elongation using homopolymeric RNA-DNA and heteropolymeric DNA-DNA complexes showed major effects of the amino acid substitutions on the Km value for dNTP. Enzymes with large hydrophobic residues at position 115 displayed lower Km values than enzymes with small and charged amino acids at this position. The influence of all these amino acid replacements in mispair extension fidelity assays was analyzed using three different mismatches (A:C, A:G and A:A) at the 3'-terminal position of the primer DNA. For the A:C mispair, a 2. 6-33.4-fold increase in mispair extension efficiency (fext) was observed as compared with the wild-type enzyme. Unexpectedly, all the mutants tested as well as the wild-type RT were very efficient in extending the A:G and A:A transversion mispairs. This effect was due to the template-primer sequence context and not to the buffer conditions of the assay. The data support a role of Tyr115 in accommodating the complementary nucleotide into the nascent DNA while polymerization takes place.

Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase

“…The reported K m for Tyr-115-Val for dTTP (62.7 M) is significantly higher than wild-type RT (6.7 M), whereas Tyr-115-Phe has a reported K m for dTTP of 3.0 M (12). Although a dTTP concentration of 10 M, which normally is used in the processivity assay, should be sufficient for wild-type RT, this concentration might be limiting for Tyr-115-Val because dTTP is present at a concentration approximately one-sixth of the reported K m value.…”

“…However, there are important differences between the results obtained with the Phe-155-Val MMLV RT variant and those obtained with the Tyr-115-Val HIV-1 RT variant. As described above, Tyr-115-Val has been reported to have a significantly higher K m for dTTP (62.7 M) than wild-type HIV-1 RT (6.7 M) (12). The difference in the dTTP K m values between wild-type MMLV RT and the MMLV RT variant is much smaller (21).…”

“…2 A and B). Tyr-115-Val has been reported to have a much higher K m for dTTP (62.7 M) than does wild-type RT (6.7 M) (12). We found that Tyr-115-Val has a higher K m for dGTP than does wild-type RT (8.61 M dGTP versus 4.50 M) and a lower V max (28.1 pmol dGTP incorporated͞min versus 38.8 pmol incorporated͞min for wild-type RT).…”

“…In analyzing mutations at Tyr-115, several groups have found that the only amino acid substitution that has little deleterious effect on the enzyme is Tyr-115-Phe (7)(8)(9)(10)(11)(12)(13)(14). Substitution of Tyr-115 with polar amino acids is extremely deleterious (9)(10)(11)(12)(13) whereas substitution with hydrophobic amino acids usually has an intermediate effect (7,8,11,12,14).…”

“…Substitution of Tyr-115 with polar amino acids is extremely deleterious (9)(10)(11)(12)(13) whereas substitution with hydrophobic amino acids usually has an intermediate effect (7,8,11,12,14). Recently, the Tyr-115-Phe mutation, along with the mutations Lys-65-Arg, Leu-74-Val, and Met-184-Val, has been detected in HIV-1 RTs resistant to the carbocyclic nucleoside analog 1592U89 (abacavir) (15,16).…”