Primosomal Proteins DnaD and DnaB Are Recruited to Chromosomal Regions Bound by DnaA inBacillus subtilis

Abstract

The initiation of DNA replication requires the binding of the initiator protein, DnaA, to specific binding sites in the chromosomal origin of replication, oriC. DnaA also binds to many sites around the chromosome, outside oriC, and acts as a transcription factor at several of these. 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 also are required to load the replicative helicase outside oriC during replication restart, independently of DnaA. 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 Bacillus subtilis. This association was dependent on DnaA, and the order of recruitment was the same as that at oriC, but it was independent of a functional oriC and suggests 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 a reservoir for excess DnaA, DnaD, and DnaB to help properly regulate replication initiation and perhaps are analogous to the proposed function of the datA locus in Escherichia 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 likely have similar functions in a range of organisms. The replication initiation protein and transcription factorDnaA is an AAAϩ ATPase that binds to many regions on the chromosome. The primary binding region is at the origin of chromosomal replication, oriC (at 0°on the B. subtilis chromosome), where there are many individual DnaA binding sites (Fig. 1). DnaA binds to oriC and causes local unwinding (melting) of the AϩT-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 references 27, 29, 49, and 56).The assembly of the replicative helicase is mediated by helicase loader proteins. In Escherichia coli, a single protein, the AAAϩ ATPase DnaC, functions to load the helicase (reviewed in references 13 and 34). In contrast, in B. subtilis and other low-GϩC Gram-positive bacteria, three different proteins, DnaD, DnaB, and the AAAϩ ATPase DnaI, are needed to load the replicative helicase (DnaC in B. subtilis) during replication initiation at oriC and 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 with oriC. 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 with oriC requires the melting of the DUE.In addition to its primary role in replication initiation and binding to sites in the oriC region, DnaA also binds to many secondary sites around the chromosome...

Chromosomal Replication Initiation Machinery of Low-G+C-Content Firmicutes

“…The hierarchy at these secondary sites is conserved, with DnaA binding first and DnaD and DnaB binding thereafter (63). Though it is possible that the recruitment of the primosomal proteins is a consequence of their functional interaction at oriC, it is equally conceivable that this may serve a regulatory role.…”

“…At least in B. subtilis, DnaD and DnaB are recruited to DnaAbinding sites outside the chromosomal origin of replication, whereas the DnaC helicase is not (63). The hierarchy at these secondary sites is conserved, with DnaA binding first and DnaD and DnaB binding thereafter (63).…”

“…These genes include some associated with tolerance to replication stress, while others are associated with the control of the sporulation cycle. The fact that DnaB and DnaD are recruited to many of these sites suggests that they may also modulate DnaA activity at the non-oriC DnaA-box clusters (63). The additional levels of control exerted by DnaB and DnaD on DnaA activity, both at the origin and elsewhere on the genome, may well increase the ability of Firmicutes such as B. subtilis to thrive in a variety of environments and respond to a wider range of conditions where E. coli cannot.…”

“…The DnaA-box cluster in the promoter region upstream of the dnaA gene also enables gene expression to be autoregulated: binding of DnaA to this DnaA-box cluster has been shown to decrease transcription and expression of the dnaA gene (50). B. subtilis has six other DnaA-box clusters (51,63), one of which is close to the dnaA gene, 3 kb upstream in the thdF-jag intergenic region (77), but they are not essential for origin function. The consensus origin organization with the three DnaA-box clusters in B. subtilis is often described as the primordial DNA replication origin.…”

DNA Repair and Genome Maintenance in Bacillus subtilis

“…Instead, B. subtilis uses components that normally function in replication initiation from oriC to fulfill the role of PriB, PriC, and DnaT in primosome assembly and replication fork restart (38,243,315). DnaD, DnaB (helicase loader in B. subtilis), and DnaI function in the loading of the B. subtilis replicative helicase DnaC at recombinational intermediates and oriC (38,243,315,393). In B. subtilis, priA-deficient strains have very slow growth and are extremely sensitive to DNA damage, and their cells filament and show aberrant nucleoid morphology, demonstrating a critical need for PriA during vegetative growth (38).…”