Bacterial origin recognition complexes direct assembly of higher-order DnaA oligomeric structures

Abstract

Eukaryotic initiator proteins form origin recognition complexes (ORCs) that bind to replication origins during most of the cell cycle and direct assembly of prereplication complexes (pre-RCs) before the onset of S phase. In the eubacterium Escherichia coli, there is a temporally similar nucleoprotein complex comprising the initiator protein DnaA bound to three high-affinity recognition sites in the unique origin of replication, oriC. At the time of initiation, this high-affinity DnaA-oriC complex (the bacterial ORC) accumulates additional DnaA that interacts with lower-affinity sites in oriC, forming a pre-RC. In this paper, we investigate the functional role of the bacterial ORC and examine whether it mediates low-affinity DnaAoriC interactions during pre-RC assembly. We report that E. coli ORC is essential for DnaA occupation of low-affinity sites. The assistance given by ORC is directed primarily to proximal weak sites and requires oligomerization-proficient DnaA. We propose that in bacteria, DnaA oligomers of limited length and stability emerge from single highaffinity sites and extend toward weak sites to facilitate their loading as a key stage of prokaryotic pre-RC assembly.R egulating chromosome duplication requires precisely timed formation of nucleoprotein complexes that comprise initiator proteins bound to replication origins and that direct assembly of new replisomes (1-6). Among the best-studied examples of such nucleoprotein complexes are the origin recognition complexes (ORCs) bound to origins in budding yeast (7,8), and the complexes formed by DnaA binding to the unique origin of chromosomal replication, oriC, in Escherichia coli (6, 9). Yeast ORC subunits share structural motifs with DnaA as well as archeal Orc1 (9, 10), and all are members of the AAAϩ family of ATPases (11). This structural conservation among initiator proteins suggests the intriguing possibility that mechanisms used by all cell types to initiate DNA synthesis could be fundamentally similar (12).Examination of the binding patterns of initiator proteins to origins during the cell cycle (5,13,14) has revealed that in addition to structural similarities, there are temporal similarities in nucleoprotein complex formation at eukaryotic and prokaryotic replication origins. Yeast ORCs bind to replication origins throughout the cell cycle and recruit additional initiator proteins needed to form the prereplicative complexes (pre-RCs) that load helicase and unwind origin DNA before entry into S phase (7,8,14,15). In E. coli, a temporally similar nucleoprotein complex is formed by DnaA binding to three high-affinity (K d Ͻ 200 nM), 9-bp recognition sites (R1, R2, and R4) within oriC (Fig. 1); like yeast ORC, this binding persists throughout the majority of the cell cycle (13,16,17), except at the time of initiation, when additional initiator DnaA binds to lower-affinity (K d Ͼ 200 nM) sites in oriC (13, 18). The additional DnaA causes localized strand separation within an AT-rich, 13-mer repeat region that is adjacent to the left side of t...

Origin Remodeling and Opening in Bacteria Rely on Distinct Assembly States of the DnaA Initiator

“…At this point, the stability of the initiator assembly likely would be compromised to some degree by the loss of DBD/AAAϩ interactions but rescued by the close proximity and phasing of duplex DnaA-binding sites, which would serve to increase the local concentration of the initiator on DNA and assist protomer association. Such behavior would help explain why ATP pro- motes the binding of DnaA to weak dsDNA sites (10,12,46,47), in that subunits situated at strong oriC loci could serve as anchor points for recruiting additional subunits to less favorable sites through nucleotide-dependent, ATPase domain contacts. Engagement of the melted DUE would then be accomplished by a different oligomer state in which the free DBD docks against a partner AAAϩ domain in trans to stabilize the DnaA assembly, rendering it competent to bind ssDNA.…”

“…During initiation, DnaA progressively renders oriC competent for replisome formation in at least three stages: (i) continual occupancy of high affinity dsDNA sites (9); (ii) cell cycle-dependent binding of additional low affinity duplex sites, along with the formation of a higher order nucleoprotein complex (10,(12)(13)(14)(15)46); and (iii) a melted state in which the DUE has been opened, likely through direct ssDNA contacts with the initiator (11,16,18). During the first two stages, we propose that the DBD extends away from the body of the initiator to expose its helix-turn-helix motif for engaging high and low affinity duplex DNA sites in oriC (Fig.…”

The Primosomal Protein DnaD Inhibits Cooperative DNA Binding by the Replication Initiator DnaA in Bacillus subtilis

“…DnaA binds both ATP and ADP, and DnaA-ATP is required for replication initiation (2,17,33,47,57). The ATP-bound form of DnaA forms oligomers that are important for promoting replication initiation (14,18,35,41,58). DnaA-ATP binds to sites in oriC and promotes the unwinding of the DNA unwinding element (DUE), which serves as a platform for assembly of the replicative helicase and the rest of the replication machinery.…”

The initiator protein DnaA, chromosomal origin oriC and their interaction to generate origin recognition complex and pre-replication complex like nucleoprotein structures in Escherichia coli

“…Aided by the helicase loader DnaC, which interacts with oriC bound ATP-DnaA, DnaB helicase is loaded onto the nucleoprotein structure, enabling subsequent bi-directional unwinding and replication fork formation 30 . It has been recently established that the propagation of DnaA protein from high affinity sites to weak affinity sites, occurs during the conversion of ORC to pre-RC 45 , Interestingly, occupation of specific sequences present in the right and left half of oriC is sequential and polarised 34 . The binding of DnaA protein emanates from R4 to R2 within the right half and assembly expands from R1 to R2 in the left half of oriC 34 .…”