The binding reactions were run on a 6% polyacrylamide gel (19:1 acrylamide/bisacrylamide) in 0.5 Tris-borate-EDTA (TBE) and 2.5% glycerol run in 0.5 TBE at approximately 12 V/cm for 3 h. suggests Mmp28 that DnaD and DnaB do not require open complex formation for the stable association with DNA. These secondary binding regions for DnaA could be serving as beta-Interleukin I (163-171), human a reservoir for excess DnaA, DnaD, and DnaB to help properly regulate replication initiation and perhaps are analogous to the proposed function of thedatAlocus inEscherichia coli. Alternatively, DnaD and DnaB might modulate the activity of DnaA at the secondary binding regions. All three of these proteins are widely conserved and beta-Interleukin I (163-171), human likely have comparable functions in a range of organisms. The replication initiation protein and transcription factor DnaA is an AAA+ ATPase that binds to many regions around the chromosome. The primary binding region is at the origin of chromosomal replication,oriC(at 0 on theB. subtilischromosome), where there are many individual DnaA binding sites (Fig.1). DnaA binds tooriCand causes local unwinding (melting) of the A+T-rich DNA unwinding element (DUE) (Fig.1) and the subsequent assembly of the replicative helicase, followed by the assembly of the rest of the replication machinery (reviewed in references27,29,49, and56). == FIG. 1. == Map of theB. subtilis oriCregion. TheoriCregions contains the clusters of DnaA binding sites upstream and downstream fromdnaAand the DNA unwinding element (DUE). The binding sites upstream of DnaA beta-Interleukin I (163-171), human contribute to the autoregulation of DnaA and also are needed fororiCfunction. Part of the DUE and the DnaA binding sites downstream fromdnaAare missing in theoriC-6mutant (1). The assembly of the replicative helicase is usually mediated by helicase loader proteins. InEscherichia coli, a single protein, the AAA+ ATPase DnaC, functions to load the helicase (reviewed in references13and34). In contrast, inB. subtilisand other low-G+C Gram-positive bacteria, three different proteins, DnaD, DnaB, and the AAA+ ATPase DnaI, are needed to load beta-Interleukin I (163-171), human the replicative helicase (DnaC inB. subtilis) during replication initiation atoriCand replication restart at stalled replication forks (5-7,13,24,40,54,55,63). There is a defined order of stable association of the replication initiation proteins withoriC. DnaA binds first, followed by DnaD and then DnaB, and finally the DnaI-mediated loading of helicase occurs (58). It is not known, however, if the association of DnaD and DnaB withoriCrequires the melting of the DUE. In addition to its primary role in replication initiation and binding to sites in theoriCregion, DnaA also binds to many secondary sites around the chromosome, away fromoriC. These secondary sites have been detected by chromatin immunoprecipitation (ChIP)-PCR, ChIP-chip, and analogous (ChAP-chip) approaches (3,18,25). Many of these secondary sites are in promoter regions, and DnaA functions as a transcription factor in several of these regions. DnaA modulates the transcription of many genes, including its own, likely under normal growth conditions and in response to replication stress and the inactivation of replication proteins (3,9,12,16,18,22,23,29,43). When replication stress is usually induced by DNA damage, the inhibition of replication elongation, or the inactivation of replication initiation protein DnaD, DnaB, or DnaI, DnaA becomes more active as a transcription factor, and the expression of target genes changes (3,9,17,18,22). DnaA activates the expression of some genes and represses the expression of others, and this apparently is dependent on the position of its binding sites relative to the RNA polymerase binding site (3,18). Because DnaD and DnaB are associated with and are recruited tooriCby DnaA, we wished to determine if they also were associated with other chromosomal regions that are bound by DnaA. We analyzed the association of DnaD and DnaB throughout theB. subtilisgenome in response to replication stress. We found that DnaD and DnaB are associated with many (perhaps all) of the chromosomal regions bound by beta-Interleukin I (163-171), human DnaA, and that there is the same dependence of association at these secondary DnaA binding regions as that atoriC: DnaA, then DnaD, then DnaB. In contrast tooriC, there was little or no detectable association of the replicative helicase (DnaC) at most of the secondary DnaA binding regions. Our findings indicate that this association of DnaD and DnaB withoriClikely does not depend around the melting of the DUE, and that there is a role for DnaD and DnaB outsideoriCand impartial of their function in replication restart. We propose that in addition to their roles in the loading of the replicative helicase, DnaD and/or DnaB is usually modulating one or more aspects of DnaA function. In addition, it is possible that the secondary DnaA binding regions are acting to modulateoriCfunction by titrating excess DnaA, DnaD, and DnaB. == MATERIALS AND METHODS == == B. subtilisstrains and alleles. == AllB. subtilisstrains (Table1) were isogenic with the laboratory wild type,.