Viral terminal protein directs early organization of phage DNA replication at the bacterial nucleoid.
The mechanism leading to protein-primed DNA replication has been studied extensively in vitro. However, little is known about the in vivo organization of the proteins involved in this fundamental process. Here we show that the terminal proteins (TPs) of phages ϕ29 and PRD1, infecting the distantly related bacteria Bacillus subtilis and Escherichia coli, respectively, associate with the host bacterial nucleoid independently of other viral-encoded proteins. Analyses of phage ϕ29 revealed that the TP N-terminal domain (residues 1-73) possesses sequence-independent DNA-binding capacity and is responsible for its nucleoid association. Importantly, we show that in the absence of the TP N-terminal domain the efficiency of ϕ29 DNA replication is severely affected. Moreover, the TP recruits the phage DNA polymerase to the bacterial nucleoid, and both proteins later are redistributed to enlarged helix-like structures in an MreB cytoskeleton-dependent way. These data disclose a key function for the TP in vivo: organizing the early viral DNA replication machinery at the cell nucleoid.
Muñoz-Espín
Daniel
D
Instituto de Biología Molecular Eladio Viñuela (Consejo Superior de Investigaciones Científicas), Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas-Universidad Autónoma), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain. dmunoz@cbm.uam.es
Holguera
Isabel
I
Ballesteros-Plaza
David
D
Carballido-López
Rut
R
Salas
Margarita
M
D017103:Bacillus Phages / Q000235:genetics / Q000502:physiology*; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism / Q000821:virology; D025622:Bacteriophage PRD1 / Q000235:genetics / Q000502:physiology*; D004261:DNA Replication / Q000235:genetics / Q000502:physiology*; D004279:DNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000821:virology; D005798:Genes, Bacterial; D005814:Genes, Viral; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics / Q000502:physiology
C054298:DNA terminal protein, Enterobacteria phage PRD1; D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C040784:terminal protein, Bacillus phage phi29; D004259:DNA-Directed DNA Polymerase
Annu Rev Biochem. 1991;60:39-71
1883199
Virology. 2007 Jul 20;364(1):237-43
17412384
Prog Nucleic Acid Res Mol Biol. 1998;60:29-46
9594570
Mol Microbiol. 1997 Sep;25(5):945-54
9364919
Mol Microbiol. 2005 Mar;55(6):1646-57
15752190
Nucleic Acids Res. 2003 Jun 15;31(12):3274-86
12799455
J Biol Chem. 2000 Dec 22;275(51):40529-38
11005822
Proc Natl Acad Sci U S A. 2008 Sep 16;105(37):14136-40
18779567
Cell. 2005 Feb 11;120(3):329-41
15707892
Genes Dev. 1990 Jul;4(7):1197-208
2210375
J Virol. 2005 Jul;79(14):9097-107
15994804
Genes Dev. 2006 Jan 1;20(1):113-24
16391237
Nucleic Acids Res. 2007;35(21):7061-73
17913744
Nucleic Acids Res. 2004 Jul 01;32(11):3493-502
15247336
J Biol Chem. 2000 May 12;275(19):14678-83
10799555
J Biol Chem. 2004 Nov 26;279(48):50437-45
15371435
J Virol. 1987 Dec;61(12):3983-91
3682063
Nucleic Acids Res. 1989 Dec 25;17(24):10353-66
2602154
Proc Natl Acad Sci U S A. 2009 Aug 11;106(32):13347-52
19654094
J Biol Chem. 2005 May 27;280(21):20730-9
15772069
BMC Cell Biol. 2005 Mar 03;6(1):10
15745453
EMBO J. 2006 Aug 23;25(16):3890-9
16888621
Curr Biol. 2003 Oct 28;13(21):1916-20
14588250
EMBO J. 2003 Oct 1;22(19):5283-92
14517265
Microbiol Mol Biol Rev. 2006 Dec;70(4):888-909
17158703
Virology. 2006 Jun 20;350(1):228-39
16530800
J Gen Microbiol. 1989 May;135(5):1335-45
2516118
Genet Eng (N Y). 1999;21:159-71
10822496
J Virol. 1993 Jun;67(6):3384-95
8497057
Mol Microbiol. 2004 Apr;52(2):529-40
15066038
Curr Top Microbiol Immunol. 2003;272:187-211
12747551
EMBO J. 2006 Mar 22;25(6):1335-43
16511564