2024-03-28T21:29:49Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/378232022-06-30T11:41:20Zcom_10261_79com_10261_1col_10261_332
DIGITAL.CSIC
author
Pérez-Arnáiz, Patricia
author
Lázaro, José M.
author
Salas, Margarita
author
Vega, Miguel de
funder
Ministerio de Ciencia e Innovación (España)
funder
Comunidad de Madrid
funder
Fundación Ramón Areces
2011-07-18T12:05:54Z
2011-07-18T12:05:54Z
2009
Journal of Molecular Biology 391(5): 797-807 (2009)
http://hdl.handle.net/10261/37823
10.1016/j.jmb.2009.06.068
http://dx.doi.org/10.13039/501100004837http://dx.doi.org/10.13039/100008054http://dx.doi.org/10.13039/100012818
Recent crystallographic resolution of 29 DNA polymerase complexes with ssDNA at its 3′-5′ exonuclease active site has allowed the identification of residues Pro129 and Tyr148 as putative ssDNA ligands, the latter being conserved in the Kx2h motif of proofreading family B DNA polymerases. Single substitution of 29 DNA polymerase residue Tyr148 to Ala rendered an enzyme with a reduced capacity to stabilize the binding of the primer terminus at the 3′-5′ exonuclease active site, not having a direct role in the catalysis of the reaction. Analysis of the 3′-5′ exonuclease on primer/template structures showed a critical role for residue Tyr148 in the proofreading of DNA polymerisation errors. In addition, Tyr148 is not involved in coupling polymerisation to strand displacement in contrast to the catalytic residues responsible for the exonuclease reaction, its role being restricted to stabilisation of the frayed 3′ terminus at the exonuclease active site. Altogether, the results lead us to extend the consensus sequence of the above motif of proofreading family B DNA polymerases into Kx2hxA. The different solutions adopted by proofreading DNA polymerases to stack the 3′ terminus at the exonuclease site are discussed. In addition, the results obtained with mutants at 29 DNA polymerase residue Pro129 allow us to rule out a functional role as ssDNA ligand for this residue.
eng
closedAccess
ø29 DNA polymerase
3′-5′ exonuclease
Site-directed mutagenesis
ssDNA binding
Functional importance of bacteriophage 29 DNA polymerase residue Tyr148 in primer-terminus stabilisation at the 3’-5’ exonuclease active site
artículo
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