Dual role of φ29 DNA polymerase Lys529 in stabilisation of the DNA priming-terminus and the terminal protein-priming residue at the polymerisation site.

Resolution of the crystallographic structure of φ29 DNA polymerase binary and ternary complexes showed that residue Lys529, located at the C-terminus of the palm subdomain, establishes contacts with the 3' terminal phosphodiester bond. In this paper, site-directed mutants at this Lys residue we...

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Autores principales:	Alicia del Prado, José M Lázaro, Laurentino Villar, Margarita Salas, Miguel de Vega
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a798d5b14b2b46c1acfe21a08e36d44c
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spelling	oai:doaj.org-article:a798d5b14b2b46c1acfe21a08e36d44c2021-11-18T08:57:01ZDual role of φ29 DNA polymerase Lys529 in stabilisation of the DNA priming-terminus and the terminal protein-priming residue at the polymerisation site.1932-620310.1371/journal.pone.0072765https://doaj.org/article/a798d5b14b2b46c1acfe21a08e36d44c2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24023769/?tool=EBIhttps://doaj.org/toc/1932-6203Resolution of the crystallographic structure of φ29 DNA polymerase binary and ternary complexes showed that residue Lys529, located at the C-terminus of the palm subdomain, establishes contacts with the 3' terminal phosphodiester bond. In this paper, site-directed mutants at this Lys residue were used to analyse its functional importance for the synthetic activities of φ29 DNA polymerase, an enzyme that starts linear φ29 DNA replication using a terminal protein (TP) as primer. Our results show that single replacement of φ29 DNA polymerase residue Lys529 by Ala or Glu decreases the stabilisation of the primer-terminus at the polymerisation active site, impairing both the insertion of the incoming nucleotide when DNA and TP are used as primers and the translocation step required for the next incoming nucleotide incorporation. In addition, combination of the DNA polymerase mutants with a TP derivative at residue Glu233, neighbour to the priming residue Ser232, leads us to infer a direct contact between Lys529 and Glu233 for initiation of TP-DNA replication. Altogether, the results are compatible with a sequential binding of φ29 DNA polymerase residue Lys529 with TP and DNA during replication of TP-DNA.Alicia del PradoJosé M LázaroLaurentino VillarMargarita SalasMiguel de VegaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 9, p e72765 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Alicia del Prado José M Lázaro Laurentino Villar Margarita Salas Miguel de Vega Dual role of φ29 DNA polymerase Lys529 in stabilisation of the DNA priming-terminus and the terminal protein-priming residue at the polymerisation site.
description	Resolution of the crystallographic structure of φ29 DNA polymerase binary and ternary complexes showed that residue Lys529, located at the C-terminus of the palm subdomain, establishes contacts with the 3' terminal phosphodiester bond. In this paper, site-directed mutants at this Lys residue were used to analyse its functional importance for the synthetic activities of φ29 DNA polymerase, an enzyme that starts linear φ29 DNA replication using a terminal protein (TP) as primer. Our results show that single replacement of φ29 DNA polymerase residue Lys529 by Ala or Glu decreases the stabilisation of the primer-terminus at the polymerisation active site, impairing both the insertion of the incoming nucleotide when DNA and TP are used as primers and the translocation step required for the next incoming nucleotide incorporation. In addition, combination of the DNA polymerase mutants with a TP derivative at residue Glu233, neighbour to the priming residue Ser232, leads us to infer a direct contact between Lys529 and Glu233 for initiation of TP-DNA replication. Altogether, the results are compatible with a sequential binding of φ29 DNA polymerase residue Lys529 with TP and DNA during replication of TP-DNA.
format	article
author	Alicia del Prado José M Lázaro Laurentino Villar Margarita Salas Miguel de Vega
author_facet	Alicia del Prado José M Lázaro Laurentino Villar Margarita Salas Miguel de Vega
author_sort	Alicia del Prado
title	Dual role of φ29 DNA polymerase Lys529 in stabilisation of the DNA priming-terminus and the terminal protein-priming residue at the polymerisation site.
title_short	Dual role of φ29 DNA polymerase Lys529 in stabilisation of the DNA priming-terminus and the terminal protein-priming residue at the polymerisation site.
title_full	Dual role of φ29 DNA polymerase Lys529 in stabilisation of the DNA priming-terminus and the terminal protein-priming residue at the polymerisation site.
title_fullStr	Dual role of φ29 DNA polymerase Lys529 in stabilisation of the DNA priming-terminus and the terminal protein-priming residue at the polymerisation site.
title_full_unstemmed	Dual role of φ29 DNA polymerase Lys529 in stabilisation of the DNA priming-terminus and the terminal protein-priming residue at the polymerisation site.
title_sort	dual role of φ29 dna polymerase lys529 in stabilisation of the dna priming-terminus and the terminal protein-priming residue at the polymerisation site.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/a798d5b14b2b46c1acfe21a08e36d44c
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_version_	1718421158963970048

Dual role of φ29 DNA polymerase Lys529 in stabilisation of the DNA priming-terminus and the terminal protein-priming residue at the polymerisation site.

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