Mutational analysis reveals <i>Escherichia coli oriC</i> interacts with both DnaA‐ATP and DnaA‐ADP during pre‐RC assembly

Grimwade, Julia E.; Torgue, Julien; McGarry, Kevin C.; Rozgaja, Tanya; Enloe, Sareena T.; Leonard, Alan C.

doi:10.1111/j.1365-2958.2007.05930.x

Cited by 37 publications

(54 citation statements)

References 59 publications

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“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Discussionmentioning

confidence: 99%

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

Duderstadt

Mott

Crisona

et al. 2010

Journal of Biological Chemistry

134

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The initiation of DNA replication requires the melting of chromosomal origins to provide a template for replisomal polymerases. In bacteria, the DnaA initiator plays a key role in this process, forming a large nucleoprotein complex that opens DNA through a complex and poorly understood mechanism. Using structure-guided mutagenesis, biochemical, and genetic approaches, we establish an unexpected link between the duplex DNA-binding domain of DnaA and the ability of the protein to both self-assemble and engage singlestranded DNA in an ATP-dependent manner. Intersubunit cross-talk between this domain and the DnaA ATPase region regulates this link and is required for both origin unwinding in vitro and initiator function in vivo. These findings indicate that DnaA utilizes at least two different oligomeric conformations for engaging single-and double-stranded DNA, and that these states play distinct roles in controlling the progression of initiation.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Duderstadt

Mott

Crisona

et al. 2010

Journal of Biological Chemistry

134

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“…Because the pBR322 origin requires DNA polymerase I to replicate, colonies arise only when plasmid oriC is functional. If the plasmid oriC harbors a mutation that makes initiation less efficient, then it will not be able to compete effectively with the chromosomal copy of oriC for initiation factors, and the plasmid will not be capable of transforming cells (25,26). In the competition assay, plasmids harboring oriC 1,2,4/R5M were not able to transform polA strains, indicating that the function of the mutant oriC was impaired sufficiently to prevent it from competing with WT chromosomal oriC (transformation efficiencies were less than 0.1% of those obtained by using WT oriC plasmids).…”

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confidence: 99%

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

Miller

Grimwade

Betteridge

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…Of the lower affinity sites, I sites (I1, I2, and I3) (87) and tau sites (τ1 and τ2) (124) clearly discriminate between nucleotide forms of DnaA, showing a 4-fold preference for DnaA-ATP (124, 165). There are conflicting data regarding binding of DnaA-ADP to R5M and R3; DMS footprinting studies indicated that these two sites have no preference for a particular DnaA nucleotide form (88, 165), while Dnase I footprinting experiments suggested that all low affinity sites, as well as R2, preferred to bind DnaA-ATP (124). The reason for these differing results is not clear, but may reflect a difference in the sensitivity of the methods used to examine binding.…”

Section: Factors Involved In Initiation Of Chromosome Replicationmentioning

confidence: 99%

Initiation of DNA Replication

Leonard

Grimwade

2010

EcoSal Plus

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In recent years it has become clear that complex regulatory circuits control the initiation step of DNA replication by directing the assembly of a multicomponent molecular machine (the orisome) that separates DNA strands and loads replicative helicase at oriC , the unique chromosomal origin of replication. This chapter discusses recent efforts to understand the regulated protein-DNA interactions that are responsible for properly timed initiation of chromosome replication. It reviews information about newly identified nucleotide sequence features within Escherichia coli oriC and the new structural and biochemical attributes of the bacterial initiator protein DnaA. It also discusses the coordinated mechanisms that prevent improperly timed DNA replication. Identification of the genes that encoded the initiators came from studies on temperature-sensitive, conditional-lethal mutants of E. coli , in which two DNA replication-defective phenotypes, "immediate stop" mutants and "delayed stop" mutants, were identified. The kinetics of the delayed stop mutants suggested that the defective gene products were required specifically for the initiation step of DNA synthesis, and subsequently, two genes, dnaA and dnaC , were identified. The DnaA protein is the bacterial initiator, and in E. coli , the DnaC protein is required to load replicative helicase. Regulation of DnaA accessibility to oriC , the ordered assembly and disassembly of a multi-DnaA complex at oriC , and the means by which DnaA unwinds oriC remain important questions to be answered and the chapter discusses the current state of knowledge on these topics.

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Mutational analysis reveals Escherichia coli oriC interacts with both DnaA‐ATP and DnaA‐ADP during pre‐RC assembly

Cited by 37 publications

References 59 publications

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

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

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

Initiation of DNA Replication

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