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.
abstract: 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.
authors.affiliation: Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma, Cantoblanco, Madrid, Spain.
authors.forename: Alicia
authors.initials: A
authors.lastname: del Prado
authors.forename: José M
authors.initials: JM
authors.lastname: Lázaro
authors.forename: Laurentino
authors.initials: L
authors.lastname: Villar
authors.forename: Margarita
authors.initials: M
authors.lastname: Salas
authors.forename: Miguel
authors.initials: M
authors.lastname: de Vega
mesh_terms: D004261:DNA Replication / Q000235:genetics / Q000502:physiology; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
chemical_list: D004259:DNA-Directed DNA Polymerase; D008239:Lysine
references.citation: Methods Enzymol. 1995;262:283-94
references.pmid: 8594354
references.citation: J Mol Biol. 2000 Jun 2;299(2):447-62
references.pmid: 10860752
references.citation: Proc Natl Acad Sci U S A. 2010 Sep 21;107(38):16548-53
references.pmid: 20823229
references.citation: Cell. 1985 Dec;43(2 Pt 1):439-48
references.pmid: 4075400
references.citation: Nucleic Acids Res. 2006;34(20):6051-63
references.pmid: 17071961
references.citation: J Biol Chem. 1989 May 25;264(15):8935-40
references.pmid: 2498321
references.citation: Cell. 1989 Oct 6;59(1):219-28
references.pmid: 2790959
references.citation: J Biol Chem. 2004 Oct 1;279(40):42018-25
references.pmid: 15292168
references.citation: Methods Enzymol. 1995;262:42-9
references.pmid: 8594366
references.citation: Mol Cell. 2004 Nov 19;16(4):609-18
references.pmid: 15546620
references.citation: EMBO J. 1996 Jul 1;15(13):3430-41
references.pmid: 8670845
references.citation: Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9579-83
references.pmid: 1409668
references.citation: J Mol Biol. 1996 Dec 6;264(3):453-64
references.pmid: 8969297
references.citation: Cell. 1997 Jun 27;89(7):1087-99
references.pmid: 9215631
references.citation: Proc Natl Acad Sci U S A. 1985 Oct;82(19):6404-8
references.pmid: 3863101
references.citation: Annu Rev Biochem. 1991;60:39-71
references.pmid: 1883199
references.citation: Nucleic Acids Res. 2012 May;40(9):3886-97
references.pmid: 22210885
references.citation: Proc Natl Acad Sci U S A. 2011 Nov 15;108(46):18655-60
references.pmid: 22065756
references.citation: EMBO J. 2007 Jul 25;26(14):3494-505
references.pmid: 17611604
references.citation: Proc Natl Acad Sci U S A. 2008 Nov 25;105(47):18290-5
references.pmid: 19011105
references.citation: J Mol Biol. 1977 Feb 15;110(1):119-46
references.pmid: 845942
references.citation: J Mol Biol. 1998 Jun 19;279(4):807-22
references.pmid: 9642062
references.citation: J Biol Chem. 1996 Apr 12;271(15):8509-12
references.pmid: 8621470
references.citation: Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3600-5
references.pmid: 10097083
references.citation: J Biol Chem. 1998 Oct 30;273(44):28966-77
references.pmid: 9786901
references.citation: Nucleic Acids Res. 2007;35(21):7061-73
references.pmid: 17913744
references.citation: J Mol Biol. 2003 Aug 22;331(4):781-94
references.pmid: 12909010
references.citation: Genet Eng (N Y). 1999;21:159-71
references.pmid: 10822496
references.citation: J Biol Chem. 1994 Nov 25;269(47):30030-8
references.pmid: 7962004
references.citation: EMBO J. 1997 May 1;16(9):2519-27
references.pmid: 9171364
references.citation: Proc Natl Acad Sci U S A. 2005 May 3;102(18):6407-12
references.pmid: 15845765
references.citation: Structure. 1999 Oct 15;7(10):1189-99
references.pmid: 10545321
references.citation: EMBO J. 1996 Mar 1;15(5):1182-92
references.pmid: 8605889
references.citation: Nucleic Acids Res. 2006 Jun 06;34(10):3107-15
references.pmid: 16757576
references.citation: Nucleic Acids Res. 1993 Aug 11;21(16):3725-30
references.pmid: 8367287
references.citation: J Mol Biol. 1996 Jul 19;260(3):369-77
references.pmid: 8757800
references.citation: EMBO J. 2006 Mar 22;25(6):1335-43
references.pmid: 16511564
references.citation: EMBO J. 1994 Dec 1;13(23):5786-92
references.pmid: 7988575