title: The β-sliding clamp directs the localization of HdaA to the replisome in Caulobacter crescentus.
abstract: The initiation of chromosome replication is tightly regulated in bacteria to ensure that it takes place only once per cell cycle. In many proteobacteria, this process requires the ATP-bound form of the DnaA protein. The regulatory inactivation of DnaA (RIDA) facilitates the conversion of DnaA-ATP into replication-inactive DnaA-ADP, thereby preventing overinitiation. Homologues of the HdaA protein, together with the β-clamp of the DNA polymerase (DnaN), are required for this process. Here, we used fluorescence resonance energy transfer experiments to demonstrate that HdaA interacts with DnaN in live Caulobacter crescentus cells. We show that a QFKLPL motif in the N-terminal region of HdaA is required for this interaction and that this motif is also needed to recruit HdaA to the subcellular location occupied by the replisome during DNA replication. An HdaA mutant protein that cannot colocalize or interact with DnaN can also not support the essential function of HdaA. These results suggest that the recruitment of HdaA to the replisome is needed during RIDA in C. crescentus, probably as a means to sense whether chromosome replication has initiated before DnaA becomes inactivated. In addition, we show that a conserved R145 residue located in the AAA+ domain of HdaA is also needed for the function of HdaA, although it does not affect the interaction of HdaA with DnaN in vivo. The AAA+ domain of HdaA may therefore be required during RIDA after the initial recruitment of HdaA to the replisome by DnaN.
authors: Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, Quartier UNIL/Sorge, Lausanne, CH 1015, Switzerland.
authors: Carmen
authors: C
authors: Fernandez-Fernandez
authors: Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.
authors: Karin
authors: K
authors: Grosse
authors: Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.
authors: Victor
authors: V
authors: Sourjik
authors: Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, Quartier UNIL/Sorge, Lausanne, CH 1015, Switzerland.
authors: Justine
authors: J
authors: Collier
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D016935:Caulobacter crescentus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004259:DNA-Directed DNA Polymerase; C083980:dnaN protein, Bacteria; D004265:DNA Helicases
references: Cell. 1994 May 6;77(3):413-26
references: 8011018
references: J Bacteriol. 1997 Feb;179(3):592-600
references: 9006009
references: J Biol Chem. 2005 Feb 25;280(8):6528-36
references: 15611053
references: EMBO J. 1999 Dec 1;18(23):6642-52
references: 10581238
references: Genes Cells. 2004 Jun;9(6):509-22
references: 15189445
references: Cell. 1998 Jul 10;94(1):61-71
references: 9674428
references: Nat Rev Mol Cell Biol. 2002 Nov;3(11):826-35
references: 12415300
references: Trends Cell Biol. 2011 Mar;21(3):188-94
references: 21123069
references: J Bacteriol. 2009 Sep;191(18):5706-16
references: 19633089
references: Genes Dev. 1998 Oct 1;12(19):3032-43
references: 9765205
references: Proc Natl Acad Sci U S A. 2002 Jan 8;99(1):123-7
references: 11742065
references: Crit Rev Biochem Mol Biol. 2008 May-Jun;43(3):163-87
references: 18568846
references: FEMS Microbiol Rev. 2002 Nov;26(4):355-74
references: 12413665
references: J Biol Chem. 2008 Dec 26;283(52):36118-31
references: 18977760
references: Cell. 1995 Sep 22;82(6):927-36
references: 7553853
references: Microbiol Mol Biol Rev. 2010 Mar;74(1):13-41
references: 20197497
references: J Biol Chem. 2006 Apr 21;281(16):10653-6
references: 16421093
references: Genome Res. 1999 Jan;9(1):27-43
references: 9927482
references: Plasmid. 2012 Mar;67(2):76-87
references: 22227374
references: J Bacteriol. 1999 Apr;181(7):1984-93
references: 10094673
references: PLoS One. 2011;6(10):e26028
references: 22022497
references: Mol Syst Biol. 2009;5:238
references: 19156130
references: Mol Microbiol. 2009 Apr;72(1):139-54
references: 19220749
references: Mol Cell Biol. 2007 Feb;27(4):1394-406
references: 17145765
references: Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347-51
references: 7012838
references: J Bacteriol. 1977 Oct;132(1):294-301
references: 334726
references: Methods Enzymol. 2000;327:472-500
references: 11045004
references: EMBO J. 1995 Aug 15;14(16):4083-9
references: 7664748
references: J Biol Chem. 1995 Apr 21;270(16):9265-71
references: 7721846
references: Proc Natl Acad Sci U S A. 2013 Jan 15;110(3):936-41
references: 23277577
references: Nat Rev Microbiol. 2007 May;5(5):343-54
references: 17435790
references: Proc Natl Acad Sci U S A. 1998 Jan 6;95(1):120-5
references: 9419339
references: J Struct Biol. 2004 Apr-May;146(1-2):106-12
references: 15095758
references: Mol Cell. 2006 Nov 17;24(4):581-92
references: 17114060
references: EMBO J. 1999 Nov 1;18(21):6169-76
references: 10545126
references: EMBO J. 2001 Sep 3;20(17):4952-63
references: 11532959
references: J Bacteriol. 2008 Aug;190(15):5368-81
references: 18502852
references: J Bacteriol. 2003 Mar;185(6):1817-24
references: 12618445
references: Annu Rev Microbiol. 2011;65:19-35
references: 21639790
references: Mol Microbiol. 2001 Apr;40(2):485-97
references: 11309130
references: Mol Microbiol. 2011 Oct;82(2):312-26
references: 21843309
references: Mol Microbiol. 2010 Jun;76(6):1555-71
references: 20487274
references: EMBO J. 2006 Mar 8;25(5):1137-47
references: 16482221
references: Nucleic Acids Res. 2007;35(20):e137
references: 17959646
references: J Bacteriol. 2002 Apr;184(8):2155-66
references: 11914347
references: Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8376-80
references: 11459978
references: Genes Dev. 2006 Aug 1;20(15):2121-34
references: 16882985
references: Curr Biol. 2011 Jul 12;21(13):1092-101
references: 21683595
references: EMBO J. 2006 Mar 8;25(5):1126-36
references: 16482215
references: Methods Enzymol. 1991;204:372-84
references: 1658564
references: J Bacteriol. 2004 Jun;186(11):3508-15
references: 15150238
references: EMBO J. 2001 Aug 1;20(15):4253-62
references: 11483528
references: Proc Natl Acad Sci U S A. 2001 Sep 25;98(20):11627-32
references: 11573000
references: Mol Microbiol. 2012 Aug;85(4):648-68
references: 22716942
references: Nat Rev Microbiol. 2010 Mar;8(3):163-70
references: 20157337
references: Mol Microbiol. 2010 Apr;76(2):302-17
references: 20132442
references: Cell. 1997 Aug 8;90(3):415-24
references: 9267022
references: Curr Protoc Protein Sci. 2001 May;Chapter 19:Unit19.5
references: 18429146
references: EMBO Rep. 2005 Aug;6(8):736-41
references: 16041320