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<i>In Vitro</i> Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA

By Janet L. Smith (213446) and Alan D. Grossman (237649)

Abstract

<div><p>DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of <i>Bacillus subtilis</i> DnaA to genomic DNA <i>in vitro</i> at single nucleotide resolution using <i>in vitro</i> DNA affinity purification and deep sequencing (IDAP-Seq). We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA <i>in vivo</i>, suggesting that other cellular factors or the amount of available DnaA <i>in vivo</i> restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA <i>in vivo</i> but not <i>in vitro</i>, and that the nucleoid-associated protein Rok was required for binding <i>in vivo</i>. Our <i>in vitro</i> characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on <i>in vivo</i> binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.</p></div

Topics: Biological Sciences, aaa, Transcription Factor DnaA DnaA, atp, DNA binding proteins, vivo, Genome DNA Binding Analysis, DNA affinity purification, DnaA binding site, Bacillus subtilis DnaA, chromosomal regions, replication initiation protein, nucleotide resolution, 269 binding regions, Bacterial Replication Initiator
Year: 2015
DOI identifier: 10.1371/journal.pgen.1005258
OAI identifier: oai:figshare.com:article/1429679
Provided by: FigShare
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