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  • Each mixture was transferred to a Poly-Prep column (Bio-Rad, Hercules, CA), and washed three times with 1 ml equilibration/wash buffer, with care taken to ensure that all washes had been accomplished beneath virtually identical situations. Complexes of DnaA-his bound to DNA have been eluted by adding 0.five ml ChIP elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1 SDS), capping the bottoms and covering the tops of your columns tightly with foil, and incubating at 65 for 15 min. The eluate was collected, as well as the resin was washed twice with 200 l ChIP elution buffer to recover all the eluted DNA. The recovered DNA was purified employing a QiaQuick PCR purification kit (Qiagen).DNA sequencingSample preparation, such as incorporation of a 3' barcode, collection of 20000 bp fragments (just after addition of adaptors and amplification), and single read sequencing (40 nt) on an Illumina HiSeq have been performed by the MIT BioMicro Center.Seq information processing and peak calling algorithmAlignment of DNA fragments bound by DnaA-his to the genome of AG1839 (a.k.a., KPL69; GenBank accession number CP008698) [29] was performed applying Bowtie [45], with adjustments to compensate for the fact that the chromosome is circular. Peak calling around the 1.four M and 4.1 M ATP-DnaA-his data was completed using cisGenome v. 2.0 [46], and in some cases PeakSplitter [47], and visualized within the genome browser MochiView [48] for manual refinement (see S1 Text for facts). The genome position in the summit of every single peak was determined using information from the 4.1 M ATP-DnaA-his binding reaction, since the peaks (especially the weaker ones) had been far better defined at this DnaA concentration. Seq data are offered at NCBI beneath accession SRX648534.Quantitation of binding and determination of apparent binding constantsTo figure out the quantity of DNA bound by DnaA-his for every chromosomal area, we determined the number of sequence reads across that area. Every sequence study (mapped to the chromosome utilizing Bowtie) was computationally extended by the estimated typical fragment length of 250 base pairs (presented schematically in S3A and S3B Fig). The relative coverage at every single bp along the chromosome was obtained by summing the Title Loaded From File amount of fragments on both the positive and negative strands that are inferred to span that position (S3C Fig). Custom R scripts were made use of for these steps. The resulting coverage map allowed distinct regions along the chromosome to be compared for any provided sample. To compare individual loci under several different binding circumstances (e.g., ATP v. ADP, or at different concentrations of DnaA), we normalized the amount of sequence reads (coveragePLOS Genetics | DOI:ten.1371/journal.pgen.Might 28,15 /Whole Genome Analysis of DNA Binding by DnaA In Vitromap amplitudes) towards the total amount of DNA recovered in each reaction (S1 Text, S3D 3G Fig). The quantity of DNA that was recovered in each and every sample increased with increasing amounts DnaA (S3F Fig), as a consequence of 1) increases in background binding, two) increases in binding at regions that have not however reached saturation, and three) binding at new weaker binding regions. Measurements in the maximum binding following normalization vs. DNA concentration gave information that might be match to a binding curve (S3H Fig).