News Column

Reports Outline DNA Research Study Results from Autonomous University

June 17, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- Investigators publish new report on DNA Research. According to news reporting originating in Madrid, Spain, by NewsRx journalists, research stated, "Tyr-226 and Tyr-390 in the 29 DNA polymerase active site are implicated in the mechanism of translocation. Y226F and Y390F differ in their effects on translocation and on dNTP and pyrophosphate binding."

The news reporters obtained a quote from the research from Autonomous University, "Mutations in the 29 DNA polymerase and exonuclease active sites perturb dNTP or pyrophosphate binding rates. DNA polymerase architecture is finely tuned to integrate translocation and substrate binding. The phi 29 DNA polymerase (DNAP) is a processive B-family replicative DNAP. Fluctuations between the pre-translocation and post-translocation states can be quantified from ionic current traces, when individual phi 29 DNAP-DNA complexes are held atop a nanopore in an electric field. Based upon crystal structures of the phi 29 DNAP-DNA binary complex and the phi 29 DNAP-DNA-dNTP ternary complex, residues Tyr-226 and Tyr-390 in the polymerase active site were implicated in the structural basis of translocation. Here, we have examined the dynamics of translocation and substrate binding in complexes formed with the Y226F and Y390F mutants. The Y226F mutation diminished the forward and reverse rates of translocation, increased the affinity for dNTP in the post-translocation state by decreasing the dNTP dissociation rate, and increased the affinity for pyrophosphate in the pre-translocation state. The Y390F mutation significantly decreased the affinity for dNTP in the post-translocation state by decreasing the association rate approximate to 2-fold and increasing the dissociation rate approximate to 10-fold, implicating this as a mechanism by which this mutation impedes DNA synthesis. The Y390F dissociation rate increase is suppressed when complexes are examined in the presence of Mn2+ rather than Mg2+. The same effects of the Y226F or Y390F mutations were observed in the background of the D12A/D66A mutations, located in the exonuclease active site, approximate to 30 from the polymerase active site."

According to the news reporters, the research concluded: "Although translocation rates were unaffected in the D12A/D66A mutant, these exonuclease site mutations caused a decrease in the dNTP dissociation rate, suggesting that they perturb phi 29 DNAP interdomain architecture."

For more information on this research see: Dynamics of Translocation and Substrate Binding in Individual Complexes Formed with Active Site Mutants of Phi 29 DNA Polymerase. Journal of Biological Chemistry, 2014;289(10):6350-6361. Journal of Biological Chemistry can be contacted at: Amer Soc Biochemistry Molecular Biology Inc, 9650 Rockville Pike, Bethesda, MD 20814-3996, USA. (American Society for Biochemistry and Molecular Biology - www.asbmb.org; Journal of Biological Chemistry - www.jbc.org/)

Our news correspondents report that additional information may be obtained by contacting J.M. Dahl, Autonomous University of Madrid, Inst Biol Mol Eladio Vinuela, CSIC, Center Biol Mol Severo Ochoa, E-28049 Madrid, Spain. Additional authors for this research include H.Y. Wang, J.M. Lazaro, M. Salas and K.R. Lieberman (see also DNA Research).

Keywords for this news article include: Spain, Madrid, Europe, Esterases, Polymerase, DNA Research, Exonucleases, Enzymes and Coenzymes

Our reports deliver fact-based news of research and discoveries from around the world. Copyright 2014, NewsRx LLC


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Source: Life Science Weekly


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