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http://dspace.mediu.edu.my:8181/xmlui/handle/10261/4925| Title: | Mechanistic Insights into the Role of Val75 of HIV-1 Reverse Transcriptase in Misinsertion and Mispair Extension Fidelity of DNA Synthesis |
| Authors: | Ministerio de Educación y Ciencia (España) Fondo de Investigación Sanitaria (España) Fundación Ramón Areces |
| Keywords: | HIV DNA polymerase Fidelity Reverse transcriptase Drug resistance |
| Publisher: | Elsevier |
| Description: | The side chain of Val75 stabilizes the fingers subdomain of the human immunodeficiency virus type 1 reverse transcriptase (RT), while its peptide backbone interacts with the single-stranded DNA template (at nucleotide + 1) and with the peptide backbone of Gln151. Specific DNA polymerase activities of mutant RTs bearing amino acid substitutions at position 75 (i.e., V75A, V75F, V75I, V75L, V75M, V75S and V75T) were relatively high. Primer extension experiments carried out in the absence of one deoxyribonucleoside-triphosphate suggested that mutations did not affect the accuracy of the RT, except for V75A, V75F, V75I, and to a lesser extent V75T. The fidelity of RTs bearing mutations V75F and V75I increased 1.8- and 3-fold, respectively, as measured by the M13 lacZ forward mutation assay, while V75A showed 1.4-fold decreased accuracy. Steady- and pre-steady-state kinetics demonstrated that the increased fidelity of V75I and V75F was related to their decreased ability to extend mismatched template–primers, while misincorporation efficiencies were not significantly affected by mutations. The increased mispair extension fidelity of mutant V75I RT could be attributed to the nucleotide affinity loss, observed in reactions with mismatched template–primers. Altogether, these data suggest that Val75 interactions with the 5′ template overhang are important determinants of fidelity Work in Madrid was supported in part by grants of the Spanish Ministry of Education and Science (BIO2003/01175), Fondo de Investigación Sanitaria (through “Red Temática de Investigación Cooperativa en SIDA” RD06/0006), and an institutional grant of Fundación Ramón Areces. T.M. is a recipient of a predoctoral fellowship of the Spanish Ministry of Education and Science Peer reviewed |
| URI: | http://dspace.mediu.edu.my:8181/xmlui/handle/10261/4925 |
| Other Identifiers: | Journal of Molecular Biology Vol. 375, Issue 5, 1 February 2008, Pages 1234-1248 0022-2836 (Print) 1089-8638 (Online) http://hdl.handle.net/10261/4925 10.1016/j.jmb.2007.11.021 |
| Appears in Collections: | Digital Csic |
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