Alan C. Leonard

Cell cycle-specific changes in nucleoprotein complexes at a chromosomal replication origin.

Initiation of DNA synthesis is triggered by the binding of proteins to replication origins. However, little is known about the order in which specific proteins associate with origin sites during the cell cycle. We show that in cycling cells there are at least two different nucleoprotein complexes at oriC. A factor for inversion stimulation (FIS)‐bound nucleoprotein complex, present throughout the majority of the cell cycle, switches to an integration host factor (IHF)‐bound form as cells initiate DNA replication. Coincident with binding of IHF, initiator DnaA binds to its previously unoccupied R3 site. In stationary phase, a third nucleoprotein complex forms. FIS is absent and inactive oriC forms a nucleoprotein structure containing IHF that is not observed in cycling cells. We propose that interplay between FIS and IHF aids assembly of initiation nucleoprotein complexes during the cell cycle and blocks initiation at inappropriate times. This exchange of components at replication origins is reminiscent of switching between pre‐ and post‐replicative chromatin states at yeast ARS1.

DNA Replication Origins

The onset of genomic DNA synthesis requires precise interactions of specialized initiator proteins with DNA at sites where the replication machinery can be loaded. These sites, defined as replication origins, are found at a few unique locations in all of the prokaryotic chromosomes examined so far. However, replication origins are dispersed among tens of thousands of loci in metazoan chromosomes, thereby raising questions regarding the role of specific nucleotide sequences and chromatin environment in origin selection and the mechanisms used by initiators to recognize replication origins. Close examination of bacterial and archaeal replication origins reveals an array of DNA sequence motifs that position individual initiator protein molecules and promote initiator oligomerization on origin DNA. Conversely, the need for specific recognition sequences in eukaryotic replication origins is relaxed. In fact, the primary rule for origin selection appears to be flexibility, a feature that is modulated either by structural elements or by epigenetic mechanisms at least partly linked to the organization of the genome for gene expression.

Escherichia coli prereplication complex assembly is regulated by dynamic interplay among Fis, IHF and DnaA.

Initiator DnaA and DNA bending proteins, Fis and IHF, comprise prereplication complexes (pre‐RC) that unwind the Escherichia coli chromosomes origin of replication, oriC. Loss of either Fis or IHF perturbs synchronous initiation from oriC copies in rapidly growing E. coli. Based on dimethylsulphate (DMS) footprinting of purified proteins, we observed a dynamic interplay among Fis, IHF and DnaA on supercoiled oriC templates. Low levels of Fis inhibited oriC unwinding by blocking both IHF and DnaA binding to low affinity sites. As the concentration of DnaA was increased, Fis repression was relieved and IHF rapidly redistributed DnaA to all unfilled binding sites on oriC. This behaviour in vitro is analogous to observed assembly of pre‐RC in synchronized E. coli. We propose that as new DnaA is synthesized in E. coli, opposing activities of Fis and IHF ensure an abrupt transition from a repressed complex with unfilled weak affinity DnaA binding sites to a completely loaded unwound complex, increasing both the precision of DNA replication timing and initiation synchrony.

Building a bacterial orisome: emergence of new regulatory features for replication origin unwinding

Triggering new rounds of chromosomal DNA replication during the bacterial cell cycle is exquisitely regulated, ensuring both proper timing and one round per cycle stringency. A critical first step is stable unwinding of oriC, the chromosomal replication origin, by multiprotein orisome complexes comprising the AAA+ initiator DnaA and modulator proteins that bend DNA. Recently identified oriC–DnaA interactions in Escherichia coli raise important questions regarding the molecular mechanisms that regulate origin unwinding in bacteria. We describe staged binding of E. coli origin recognition proteins and suggest an unwinding switch based on interactions between DnaA‐ATP and specialized oriC sites that must be filled during orisome assembly. By focusing multiple regulatory pathways on only a few key oriC DNA–protein interactions, this model includes an efficient way to control unwinding followed by orisome inactivation during the cell cycle. Future studies will determine whether this regulatory scheme is correct and whether it is generally applicable to other bacterial types.

Regulation of DnaA Assembly and Activity: Taking Directions from the Genome

To ensure proper timing of chromosome duplication during the cell cycle, bacteria must carefully regulate the activity of initiator protein DnaA and its interactions with the unique replication origin oriC. Although several protein regulators of DnaA are known, recent evidence suggests that DnaA recognition sites, in multiple genomic locations, also play an important role in controlling assembly of pre-replicative complexes. In oriC, closely spaced high- and low-affinity recognition sites direct DnaA-DnaA interactions and couple complex assembly to the availability of active DnaA-ATP. Additional recognition sites at loci distant from oriC modulate DnaA-ATP availability by repressing new synthesis, recharging inactive DnaA-ADP, or titrating DnaA. Relying on genomic DnaA binding sites, as well as protein regulators, to control DnaA function appears to provide the best combination of high precision and dynamic regulation necessary to couple DNA replication with cell growth over a range of nutritional conditions.

IHF and HU stimulate assembly of pre‐replication complexes at Escherichia coli oriC by two different mechanisms

Pre‐replication complexes (pre‐RC) assemble on replication origins and unwind DNA in the presence of chromatin proteins. As components of Escherichia coli pre‐RC, two histone‐like proteins HU and IHF (integration host factor), stimulate initiator DnaA‐catalysed unwinding of the chromosomal replication origin, oriC. Using in vivo footprint analysis just before DNA synthesis initiates, we detect IHF binding coincident with a shift of DnaA to weaker central oriC sites. Integration host factor redistributed pre‐bound DnaA to identical sites in vitro. HU did not redistribute DnaA, but suppressed binding specifically at I3. These results suggest that different pathways mediated by bacterial chromatin proteins exist to regulate pre‐RC assembly and unwind oriC.

Correlation of gene transcription with the time of initiation of chromosome replication in Escherichia coli

Transcriptional levels of the Escherichia coli mioC and gidA genes, which flank the chromosomal origin of replication (oriC) and the dnaA gene, were correlated with the time of initiation of chromosome replication. The transcripts were measured either in dnaC2(ts) mutants that had been aligned for initiation of chromosome replication by a temperature shift or in synchronous cultures of cells obtained using the baby machine technique. In both types of experiments, mioC transcription was inhibited prior to initiation of chromosome replication and resumed several minutes after initiation. Conversely, gidA and dnaA transcription were both inhibited after initiation of replication, coincident with the period of hemimethylation of oriC DNA. It is proposed that mioC transcription prevents initiation of chromosome replication, and must terminate before replication can begin. It is further proposed that the eclipse period between rounds of replication, i.e. the minimum interval between successive initiations, encompasses the time required to methylate GATC sequences in newly replicated oriC plus the time required to terminate mioC transcription. Conversely, the active transcription of gidA and dnaA prior to initiation is consistent with their positive effects on initiation, and their shutdown after initiation could serve to limit premature reinitiation.

IHF redistributes bound initiator protein, DnaA, on supercoiled oriC of Escherichia coli

In Escherichia coli, initiation of chromosome replication requires that DnaA binds to R boxes (9‐mer repeats) in oriC, the unique chromosomal replication origin. At the time of initiation, integration host factor (IHF) also binds to a specific site in oriC. IHF stimulates open complex formation by DnaA on supercoiled oriC in cell‐free replication systems, but it is unclear whether this stimulation involves specific changes in the oriC nucleoprotein complex. Using dimethylsulphate (DMS) footprinting on supercoiled oriC plasmids, we observed that IHF redistributed prebound DnaA, stimulating binding to sites R2, R3 and R5(M), as well as to three previously unidentified non‐R sites with consensus sequence (A/T)G(G/C) (A/T)N(G/C)G(A/T)(A/T)(T/C)A. Redistribution was dependent on IHF binding to its cognate site and also required a functional R4 box. By reducing the DnaA level required to separate DNA strands and trigger initiation of DNA replication at each origin, IHF eliminates competition between strong and weak sites for free DnaA and enhances the precision of initiation synchrony during the cell cycle.

Two oppositely oriented arrays of low‐affinity recognition sites in oriC guide progressive binding of DnaA during Escherichia coli pre‐RC assembly

The onset of chromosomal DNA replication requires highly precise and reproducible interactions between initiator proteins and replication origins to assemble a pre‐replicative complex (pre‐RC) that unwinds the DNA duplex. In bacteria, initiator protein DnaA, bound to specific high‐ and low‐affinity recognition sites within the unique oriC locus, comprises the pre‐RC, but how complex assembly is choreographed to ensure precise initiation timing during the cell cycle is not well understood. In this study, we present evidence that higher‐order DnaA structures are formed at oriC when DnaA monomers are closely positioned on the same face of the DNA helix by interaction with two oppositely oriented essential arrays of closely spaced low‐affinity DnaA binding sites. As DnaA levels increase, peripheral high‐affinity anchor sites begin cooperative loading of the arrays, which is extended by sequential binding of additional DnaA monomers resulting in growth of the complexes towards the centre of oriC. We suggest that this polarized assembly of unique DnaA oligomers within oriC plays an important role in mediating pre‐RC activity and may be a feature found in all bacterial replication origins.

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

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 DnaA–oriC 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 high-affinity sites and extend toward weak sites to facilitate their loading as a key stage of prokaryotic pre-RC assembly.