Innovating Works

PGC2018-093723-A-I00

Financiado
DNA POLIMERASAS INDEPENDIENTES DE PRIMER Y SUS APLICACIONES EN BIOTECNOLOGIA Y B...
PRIMER-INDEPENDENT DNA POLYMERASES GROUP (PIPOLBS) CONSTITUTES A THIRD, PREVIOUSLY OVERLOOKED, CLADE OF FAMILY B DNA POLYMERASES (POLBS), ALONG WITH THE RNA-PRIMED (RPOLBS) AND PROTEIN-PRIMED (PPOLBS), PIPOLBS ARE ENCODED BY A NEW... PRIMER-INDEPENDENT DNA POLYMERASES GROUP (PIPOLBS) CONSTITUTES A THIRD, PREVIOUSLY OVERLOOKED, CLADE OF FAMILY B DNA POLYMERASES (POLBS), ALONG WITH THE RNA-PRIMED (RPOLBS) AND PROTEIN-PRIMED (PPOLBS), PIPOLBS ARE ENCODED BY A NEW GROUP OF MOBILE GENETIC ELEMENTS, NAMED PIPOLINS, MOST OF WHICH ARE INTEGRATED INTO GENOMES OF BACTERIA FROM PHYLA FIRMICUTES, ACTINOBACTERIA, AND PROTEOBACTERIA, BUT ALSO REPLICATING AS CIRCULAR PLASMIDS IN MITOCHONDRIA, ANALYSIS OF MULTIPLE SEQUENCE ALIGNMENTS (MSAS) OF PIPOLBS SHOWED THAT ALL OF THEM CONTAINED ACTIVE SITE RESIDUES OF THE EXONUCLEASE AND DNA POLYMERASE ACTIVITIES OF POLBS, ALBEIT WITH NOTABLE VARIATIONS WITHIN THE KXY AND POL C MOTIFS, INITIAL BIOCHEMICAL CHARACTERIZATION OF PIPOLBS SHOWED THAT THEY SHARE SOME PROPERTIES WITH OTHER REPLICATIVE DNA POLYMERASES, LIKE A PROOFREADING CAPACITY COUPLED TO PROCESSIVE AND FAITHFUL DNA POLYMERIZATION ACTIVITY, STRIKINGLY, THEY ARE ALSO CAPABLE OF PRIMER-INDEPENDENT DE NOVO DNA SYNTHESIS, I,E,, DNA-PRIMING ACTIVITY, THEREBY BREAKING THE LONG-STANDING DOGMA THAT REPLICATIVE DNA POLYMERASES REQUIRE A PRE-EXISTING PRIMER FOR DNA SYNTHESIS, A MODIFY EXTENDED KXY MOTIF, KH-X8-TGR, IS INVOLVED IN THIS RELEVANT CAPACITY,MOREOVER, PIPOLBS DO NOT DISPLAY STRONG SEQUENCE REQUIREMENT FOR REPLICATION INITIATION AND REPLICATION ORIGINS SEEM TO BE SELECTED IN A RANDOM MANNER, A PROPERTY THAT MAY BE USEFUL FOR DEVELOPMENT OF UNBIASED WHOLE-GENOME AMPLIFICATION,IN THIS PROJECT, WE PROPOSE THE COMBINED USE OF BIOINFORMATIC, GENETIC AND BIOCHEMISTRY METHODS TO ANALYZE IN DETAIL THE PROPERTIES OF PIPOLBS BY MUTAGENESIS OF CLADE-SPECIFIC CONSERVED RESIDUES, NAMELY, THE ROLE OF AN INVARIABLE ADDITIONAL ASPARTATE OF THE POL A MOTIF AND THE MODIFIED POL C MOTIF “TTDG” WILL BE DISCLOSED, A HYBRID PHYLOGENETIC ANALYSIS, USING A COMBINATION OF MSA AND STRUCTURAL COMPARISONS, WILL BE USED TO PERFORM A COMPREHENSIVE CLASSIFICATION OF POLBS IN DATABASE AND SELECT NEW CANDIDATES WITH UNFORESEEN PROPERTIES FOR BIOCHEMICAL CHARACTERIZATION, IN ADDITION, IN ORDER TO IMPROVE THE EFFICIENCY OF REPLICATION AND AMPLIFICATION ASSAYS IN VITRO, WE WILL SEEK FOR PIPOLB-INTERACTING PROTEINS THAT MAY BE REQUIRED FOR PIPOLIN REPLICATION IN VIVO,FURTHERMORE, BESIDES PROVIDING NEW INSIGHTS INTO THE EVOLUTION AND MOLECULAR MECHANISMS OF DNA REPLICATION, WE WILL CONTRIBUTE TO THE DEVELOPING OF NOVEL BIOTECHNOLOGY APPLICATIONS FOR PRIMER-INDEPENDENT DNA POLYMERASES, ANALYZING WHOLE GENOME AMPLIFICATION COVERAGE BY NEXT-GENERATION SEQUENCING METHODS AND SETTING UP MICROFLUIDICS METHODS FOR SINGLE-MOLECULE DNA AMPLIFICATION USING BOTH, PIPOLB AND NEW CANDIDATES OF INTEREST DETECTED DURING THE PROJECT, ADN POLIMERASAS\REPLICACIÓN DEL ADN\ELEMENTO GENÉTICO MÓVIL\AMPLIFICACIÓN DEL ADN\MICROFLUÍDICA ver más
01/01/2018
UAM
127K€
Perfil tecnológico estimado

Línea de financiación: concedida

El organismo AGENCIA ESTATAL DE INVESTIGACIÓN notifico la concesión del proyecto el día 2018-01-01
Presupuesto El presupuesto total del proyecto asciende a 127K€
Líder del proyecto
UNIVERSIDAD AUTÓNOMA DE MADRID No se ha especificado una descripción o un objeto social para esta compañía.
Total investigadores 3183