Mutations in the ribonuclease H active site of HIV-RT reveal a role for this site in stabilizing enzyme-primer-template binding.
The RNase H activity of HIV-RT is coordinated by a catalytic triad (E478, D443, D498) of acidic residues that bind divalent cations. We examined the effect of RNase H deficient E(478)-->Q and D(549)-->N mutations that do not alter polymerase activity on binding of enzyme to various nucleic acid substrates. Binding of the mutant and wild-type enzymes to various nucleic acid substrates was examined by determining dissociation rate constants (k(off)) by titrating both Mg(2+) and salt concentrations. In agreement with the unaltered polymerase activity of the mutant, the k(off) values for the wild-type and mutant enzymes were essentially identical using DNA-DNA templates in the presence of 6 mM Mg(2+). However, with lower concentrations of Mg(2+) and in the absence of Mg(2+), although both enzymes dissociated more rapidly, the mutant enzymes dissociated several-fold more slowly than the wild type. This was also observed on RNA-DNA templates. These results indicate that alterations in residues essential for Mg(2+) binding have a pronounced positive effect on enzyme-template stability and that the negative residues in the RNase H region of the enzyme have a negative influence on binding in the absence of Mg(2+). In this regard RT is similar to other nucleic acid cleaving enzymes that show enhanced binding upon mutation of active site residues.
Cristofaro
Jason V
JV
Department of Cell Biology and Molecular Genetics, University of Maryland College Park, Building 231, College Park, Maryland 20742, USA.
Rausch
Jason W
JW
Le Grice
Stuart F J
SF
DeStefano
Jeffrey J
JJ
D001665:Binding Sites / Q000235:genetics; D002413:Cations, Divalent / Q000737:chemistry; D017931:DNA Primers / Q000138:chemical synthesis / Q000737:chemistry*; D004795:Enzyme Stability / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics*; D006639:Histidine / Q000235:genetics; D006868:Hydrolysis; D015636:Magnesium Chloride / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D011485:Protein Binding / Q000235:genetics; D012313:RNA / Q000138:chemical synthesis / Q000737:chemistry*; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics; D012367:RNA, Viral / Q000138:chemical synthesis / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics*; D013379:Substrate Specificity / Q000235:genetics; D013698:Templates, Genetic
D002413:Cations, Divalent; D017931:DNA Primers; C034477:RNA primers; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D015636:Magnesium Chloride; D006639:Histidine; D012313:RNA; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H