Record number :
1242307
Title of article :
Substitutions at Phe61 in the β3-β4 Hairpin of HIV-1 Reverse Transcriptase Reveal a Role for the Fingers Subdomain in Strand Displacement DNA Synthesis
Author/Authors :
Timothy S Fisher، نويسنده , , Tom Darden، نويسنده , , Vinayaka R Prasad، نويسنده ,
Issue Information :
روزنامه با شماره پیاپی سال 2003
Pages :
17
From page :
443
To page :
459
Abstract :
Unlike most DNA polymerases, retroviral reverse transcriptases (RTs) are capable of strand displacement DNA synthesis in vitro, unassisted by other proteins. While human immunodeficiency virus type 1 (HIV-1) RT has been shown to possess this rare ability, the structural determinants responsible are unknown. X-Ray crystallographic and biochemical studies have indicated that the β3-β4 hairpin of the fingers subdomain of HIV-1 RT contains key contacts for the incoming template strand. In order to assess the possible role of the fingers subdomain in strand displacement synthesis, a set of substitutions was created at the highly conserved Phe61 residue, which is thought to contact the template strand immediately ahead of the dNTP-binding site. Purified heterodimeric RTs containing Phe61 substitutions displayed altered degrees of strand displacement synthesis on nicked and gapped duplex DNA templates with the relative order being: F61Y≥F61L>wild-type=F61A>F61W. In order to verify that the effects on strand displacement synthesis were not an indirect effect of alterations in processivity, all Phe61 mutants were tested for processive polymerization. While the strand displacement activity of F61W RT variant was affected severely, it displayed a wild-type-like processivity. In contrast, both F61L and F61Y substitutions, despite showing enhanced strand displacement synthesis, displayed reduced processivity. In contrast, the processivity of F61A mutant, which had displayed nearly wild-type-like strand displacement synthesis, was affected most. These results showed that the effects of Phe61 substitutions on strand displacement are not due to global changes in polymerase processivity. Analysis of pause sites during DNA polymerization on double-stranded templates revealed that the wild-type and the Phe61 mutant RTs interact with the template quite differently. Modeling a 5 nt duplex DNA ahead of the dNTP-binding site of HIV-1 RT suggested a correlation between the ability of the side-chain of the amino acid residue at position 61 to stabilize the first base-pair of the DNA duplex to be melted and the degree of strand displacement synthesis. Our results confirm a role for F61 residue in processive synthesis and indicate that the fingers subdomain harbors a structural determinant of strand displacement synthesis by HIV-1 RT.
Keywords :
Human Immunodeficiency Virus , reverse transcriptase , displacement DNA synthesis , fingers subdomain , processivity
Journal title :
Journal of Molecular Biology
Link To Document :