Comment[ArrayExpressAccession]	E-GEOD-23686
Public Release Date	2010-12-07									
Investigation Title	Genome-wide binding of replication initiation proteins in Bacillus subtilis									
Comment[Submitted Name]	Genome-wide binding of replication initiation proteins in Bacillus subtilis
Experiment Description	Initiation of DNA replication requires binding of the initiator protein, DnaA, to specific binding sites in the chromosomal origin of replication, oriC. In low G+C Gram-positive bacteria, the primosomal proteins DnaD and DnaB, in conjunction with loader ATPase DnaI, load the replicative helicase at oriC, and this depends on DnaA. DnaD and DnaB are also required to load the replicative helicase outside of oriC during replication restart, in a DnaA-independent manner. DnaA also binds to many sites around the chromosome, outside of oriC, and acts as a transcription factor at several of these. Using chromatin immunoprecipitation, we found that DnaD and DnaB, but not the replicative helicase, are associated with many of the chromosomal regions bound by DnaA in vivo in Bacillus subtilis. This association was dependent on DnaA and the order of recruitment was the same as that at oriC, but was independent of a functional oriC.  The presence of DnaD and DnaB at the secondary (non-oriC) targets of DnaA in the absence of helicase loading indicates a possible role for DnaD and DnaB in modulating the activity of DnaA. The genome-wide binding profiles of DnaA, DnaD, DnaB and DnaC were determined. Binding profiles were determined in exponentially growing cells with and without HPUra treatment. Three biological replicates were analyzed per protein/treatment (one per array). Enrichment in immunoprecipitated samples versus total genomic DNA were determined.									
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Term Source Name	EFO
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Term Source File	http://www.ebi.ac.uk/efo/efo.owl									
Person Last Name	Grossman	Smits	Merrikh	Bonilla	Grossman					
Person First Name	Alan	Wiep	Houra	Carla	Alan					
Person Mid Initials	D.	K		Y	D					
Person Email	clee2@mit.edu									
Person Affiliation	MIT									
Person Phone	617-253-6702									
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Person Address	Biology, MIT, 31 Ames Street, 68-530, Cambridge, MA, USA									
Person Roles	submitter									
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Protocol Name	P-GSE23686-1	P-GSE23686-6	P-GSE23686-2	P-GSE23686-8	P-GSE23686-7	P-GSE23686-9	P-GSE23686-5	P-GSE23686-10	P-GSE23686-3	P-GSE23686-4
Protocol Description	ID_REF = <br>VALUE = Lowess-normalized log2 ratio (IP/total)	Samples were hybridized as described in Auchtung et al. 2005 PNAS 102:12554-9 [PMID 16105942].	Cells were grown in minimal 1% glucose medium until OD600 reached approximately 0.6. At this point, cells were treated with HPUra or not, and growth was continued for another 30 minutes before harvesting.	GPR files were loaded and processed in Prep+07 (BMC Bioinformatics 2009, 10:16 [PMID 19134227; doi:10.1186/1471-2105-10-16). Lowess normalization was performed and spots for which signal was 90% within 2SD above background were removed. Results from the Prep+07 analysis were manually curated in Microsoft Excel for spots that had <2 datapoints with quality data for downstream analysis.	Arrays were scanned and analyzed with GenePix Pro 3.0 (Axon Instruments).	To induce replication stress by arresting the replicative polymerase PolC, the inhibitor HPUra was added at a final concentration of 38 ug/mL to exponentially growing cells for 30 minutes.	Chromatin immunoprecipitation (ChIP) of DNA bound to native proteins was done essentially as described (1998, Lin and Grossman, Cell, 92:675-685 [PMID 9506522]; 2007, Breier and Grossman, Molecular Microbiology, 64:703-718 [PMID 17462018]).	Chromatin immunoprecipitation (ChIP) of DNA bound to native proteins was done essentially as described (1998, Lin and Grossman, Cell, 92:675-685 [PMID 9506522]; 2007, Breier and Grossman, Molecular Microbiology, 64:703-718 [PMID 17462018]). Anti-DnaB/C/D were custom antibodies (sera), and were obtained from Covance (CRP, Inc); they do not have a catalog number. Anti-DnaB, anti-DnaC and anti-DnaD were raised in rabbits after purification of tagged proteins in the Grossman lab. For the experiments, the following bleeds were used: DnaB: rabbit HM654, exsanguination DnaC: rabbit HM6351, test bleed 2 DnaD: rabbit HM6354, production bleed 3 The IP requires a second antibody: AffiniPure Donkey Anti-Chicken IgY from Jackson ImmunoResearch Laboratories Inc. Catalog #703-005-155.	Chromatin immunoprecipitation (ChIP) of DNA bound to native proteins was done essentially as described (1998, Lin and Grossman, Cell, 92:675-685 [PMID 9506522]; 2007, Breier and Grossman, Molecular Microbiology, 64:703-718 [PMID 17462018]). Anti-DnaA was raised in chicken and comes from W. F. Burkholder (Stanford). The use of these antibodies is described in Goranov 2005 Proc Nat Acad Sci USA and Breier 2009 J. Bacteriol. Anti-DnaA serum came from chicken #1316. The IP requires a second antibody: AffiniPure Donkey Anti-Chicken IgY from Jackson ImmunoResearch Laboratories Inc. Catalog #703-005-155.	Samples were prepared for microarray analysis by labelling aliquots of the resuspended immunoprecipitated DNA or total control DNA with aminoallyl-dUTP using 13 U Sequenase (USB) and 5 μg random nonamer. The labelled samples were purified, conjugated to Cy3 or Cy5, and hybridized to microarrays.
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Protocol Type	bioassay_data_transformation	hybridization	grow	feature_extraction	image_aquisition	specified_biomaterial_action	nucleic_acid_extraction	nucleic_acid_extraction	nucleic_acid_extraction	labeling
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Experimental Factor Name	ISOLATION	TREATMENT	ANTIBODY							
Experimental Factor Type	isolation	treatment	antibody							
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Publication Title	Primosomal proteins DnaD and DnaB are recruited to chromosomal regions bound by DnaA in Bacillus subtilis.									
Publication Author List	Smits WK, Merrikh H, Bonilla CY, Grossman AD									
PubMed ID	21097613									
Publication DOI	10.1128/JB.01253-10									
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Comment[SecondaryAccession]	GSE23686									
Comment[GEOLastUpdateDate]	2012-03-22
Comment[AEExperimentType]	ChIP-chip by array									
Comment[GEOReleaseDate]	2010-12-07
Comment[ArrayExpressSubmissionDate]	2010-08-17
SDRF File	E-GEOD-23686.sdrf.txt