{"id":109464,"date":"2018-03-11T10:34:53","date_gmt":"2018-03-11T10:34:53","guid":{"rendered":"https:\/\/www.deberes.net\/tesis\/sin-categoria\/caracterizacion-bioqua%c2%admica-de-la-dna-polimerasa-x-de-bacillus-subtilis\/"},"modified":"2018-03-11T10:34:53","modified_gmt":"2018-03-11T10:34:53","slug":"caracterizacion-bioqua%c2%admica-de-la-dna-polimerasa-x-de-bacillus-subtilis","status":"publish","type":"post","link":"https:\/\/www.deberes.net\/tesis\/sin-categoria\/caracterizacion-bioqua%c2%admica-de-la-dna-polimerasa-x-de-bacillus-subtilis\/","title":{"rendered":"\u00abcaracterizaci\u00f3n bioqu\u00edmica de la dna polimerasa x de bacillus subtilis\u00bb"},"content":{"rendered":"<h2>Tesis doctoral de <strong> Benito Ba\u00f1os Pi\u00f1ero <\/strong><\/h2>\n<p>Bacillus subtilis gene yshc encodes a 64-kda family x dna polymerase (polxbs). Biochemical analysis of the purified enzyme indicates that polxbs is a strictly template directed dna polymerase, preferentially acting on dna structures containing gaps from one to a few nucleotides and bearing a phosphate group at the 5\u00c2\u00bf end of the downstream dna. In addition to the polymerization activity, polxbs possesses an intrinsic 3\u00c2\u00bf-5\u00c2\u00bf exonuclease activity specialized in resecting the unannealed 3\u00c2\u00bf-termini in a gapped dna substrate. Analysis of a polxbs deletion mutant lacking the c terminal polymerase histidinol phosphatase domain (php), present in most of the bacterial\/archaeal polx, as well as of this separately expressed protein region, allows us to state that the 3\u00c2\u00bf-5\u00c2\u00bf exonuclease activity of polxbs resides in its php domain. Furthermore, site directed mutagenesis of polxbs residues h339, h341, d346, h371, e410, h437, h465, d526 and h528, evolutionary conserved in the php superfamily members, demonstrated that the predicted metal binding site is directly involved in catalysis of the exonucleolytic reaction. These results led us to propose a potential role for polxbs in dna repair.  polxbs, along with polymerization and 3\u00c2\u00bf-5\u00c2\u00bf exonuclease activities, possesses an intrinsic ap-endonuclease activity. Both, ap-endonuclease and 3\u00c2\u00bf-5\u00c2\u00bf exonuclease activities are genetically linked and governed by the same metal ligands located at the php domain. The different catalytic functions of polxbs enable it to perform recognition and incision at an ap site and further restoration of the original nucleotide in a standalone ap-endonuclease-independent way.  family x dna polymerases posses two conserved hhh (helix-hairpin-helix) motifs involved in dna binding. However, bacterial and archaeal polx have an specific third hhh motif located at the fingers subdomain. Mutational analysis of g130, g132 and k134 residues from this motif in polxbs demonstrates a role for these residues in dna interaction, polymerization and exonuclease activities on gapped substrates, allowing us to conclude an essential function for this specific hhh motif in dna binding as well as a potential role in the proper coordination between the synthetic and degradative activities in bacterial and archaeal family x dna polymerases.  many bacterial polx have a lysine residue preceding the first two catalytic aspartates involved in metal coordination during the polymerization reaction. Substitutions of residue k192 of polxbs by conservative or non-conservative residues endow the polymerase with a strand displacement ability and improved polymerization activity. These results, together with the decrease in the 3\u00c2\u00bf-5\u00c2\u00bf exonuclease activity observed in the mutant enzymes, led us to suggest a role for this residue in stabilization of the 3\u00c2\u00bf terminus in the polymerization active site.  by size exclusion chromatography techniques, polxbs was observed to form tetramers in vitro, being the php domain mainly responsible for this phenomenon. Polxbs binds specifically and with high stability to holyday junction structures, intermediates of homologous recombination repair pathways. Therefore, it is tempting to speculate that polxbs tetramers could bind to the four-way holliday junction structure, suggesting a potential role for this polymerase in homologous recombination in b. Subtilis.<\/p>\n<p>&nbsp;<\/p>\n<h3>Datos acad\u00e9micos de la tesis doctoral \u00ab<strong>\u00abcaracterizaci\u00f3n bioqu\u00edmica de la dna polimerasa x de bacillus subtilis\u00bb<\/strong>\u00ab<\/h3>\n<ul>\n<li><strong>T\u00edtulo de la tesis:<\/strong>\u00a0 \u00abcaracterizaci\u00f3n bioqu\u00edmica de la dna polimerasa x de bacillus subtilis\u00bb <\/li>\n<li><strong>Autor:<\/strong>\u00a0 Benito Ba\u00f1os Pi\u00f1ero <\/li>\n<li><strong>Universidad:<\/strong>\u00a0 Aut\u00f3noma de Madrid<\/li>\n<li><strong>Fecha de lectura de la tesis:<\/strong>\u00a0 24\/06\/2011<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<h3>Direcci\u00f3n y tribunal<\/h3>\n<ul>\n<li><strong>Director de la tesis<\/strong>\n<ul>\n<li>Jos\u00e9 Miguel De Vega<\/li>\n<\/ul>\n<\/li>\n<li><strong>Tribunal<\/strong>\n<ul>\n<li>Presidente del tribunal: jose Fernandez piqueras <\/li>\n<li>Antonio Bernad miana (vocal)<\/li>\n<li>enrique Viguera m\u00ednguez (vocal)<\/li>\n<li>Luis Blanco d\u00e1vila (vocal)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Tesis doctoral de Benito Ba\u00f1os Pi\u00f1ero Bacillus subtilis gene yshc encodes a 64-kda family x dna polymerase (polxbs). Biochemical analysis [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"site-sidebar-layout":"default","site-content-layout":"","ast-site-content-layout":"","site-content-style":"default","site-sidebar-style":"default","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"","ast-breadcrumbs-content":"","ast-featured-img":"","footer-sml-layout":"","theme-transparent-header-meta":"","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"default","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"tablet":{"background-color":"","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"mobile":{"background-color":"","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""}},"ast-content-background-meta":{"desktop":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"tablet":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"mobile":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""}},"footnotes":""},"categories":[1],"tags":[51006,218955,64806,14602,194973,2617],"class_list":["post-109464","post","type-post","status-publish","format-standard","hentry","category-sin-categoria","tag-antonio-bernad-miana","tag-benito-banos-pinero","tag-enrique-viguera-minguez","tag-jose-fernandez-piqueras","tag-jose-miguel-de-vega","tag-luis-blanco-davila"],"_links":{"self":[{"href":"https:\/\/www.deberes.net\/tesis\/wp-json\/wp\/v2\/posts\/109464","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.deberes.net\/tesis\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.deberes.net\/tesis\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.deberes.net\/tesis\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.deberes.net\/tesis\/wp-json\/wp\/v2\/comments?post=109464"}],"version-history":[{"count":0,"href":"https:\/\/www.deberes.net\/tesis\/wp-json\/wp\/v2\/posts\/109464\/revisions"}],"wp:attachment":[{"href":"https:\/\/www.deberes.net\/tesis\/wp-json\/wp\/v2\/media?parent=109464"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.deberes.net\/tesis\/wp-json\/wp\/v2\/categories?post=109464"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.deberes.net\/tesis\/wp-json\/wp\/v2\/tags?post=109464"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}