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.
Department of Cell Biology and Molecular Genetics, University of Maryland College Park, Building 231, College Park, Maryland 20742, USA.
Jason V
JV
Cristofaro
Jason W
JW
Rausch
Stuart F J
SF
Le Grice
Jeffrey J
JJ
DeStefano
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