Initiation of DNA replication in eukaryotes, archea, and eubacteria requires interaction of structurally conserved ATP-binding initiator proteins and origin DNA to mediate assembly of replisomes. However, the specific requirement for ATP in the early steps of initiation remains unclear. This is true even for the well studied Escherichia coli replication origin, oriC, where the ATP form of initiator DnaA is necessary and sufficient for initial DNA strand separation, but the five DnaA-binding sites (R boxes) with consensus sequence 5TGTGNAT͞AAA bind both active ATP-DnaA and inactive ADP-DnaA with equal affinity. By using dimethyl sulfate footprinting, we recently identified two initiator-binding sites, I2 and I3, with sequence 5TG͞TGGATCAG͞A. We now show that sites I2 and I3 preferentially bind DnaA-ATP and are required for origin unwinding. Guanine at position 3 determines DnaA-ATP preference, and changing this base to thymine at both I sites allows DnaA-ADP to bind and open oriC, although DNA strand separation is not precisely localized in the AT-rich region. These observations indicate that specific initiator binding sites within a replication origin can be important determinants of an ATP-dependent molecular switch regulating DNA strand separation. O ne of the earliest steps in triggering new rounds of DNA synthesis requires binding of initiator proteins to replication origin DNA (1). Several origin-binding proteins in eukaryotes, archea, and eubacteria are members of the AAAϩ family of ATPases, which are active in the ATP-bound form and are inactivated by hydrolysis of ATP to ADP (1-4). These proteins form complexes that mediate subsequent steps of initiation, including DNA strand separation and recruitment of replisome components (1). The recent discovery of structural conservation in AAAϩ initiation proteins in bacteria, archea, and yeast (3-5) suggests that aspects of the initiation mechanism may be similar across all domains of life. However, additional information on the specific function of ATP in the early steps of initiation is required before common initiation mechanisms can be inferred.For a variety of bacterial chromosomal and plasmid origins, as well as some eukaryotic viral origins, the first step of initiation requires binding of multiple copies of an initiator protein that serves to melt the DNA duplex in an AT-rich region (1, 6). This process has been extensively studied in Escherichia coli, where initial strand opening requires multiple copies of the AAAϩ initiator DnaA (7,8). Only ATP-DnaA is active in formation of the open complex (8, 9). Within oriC are five 9-mer DnaAbinding sites (R boxes; see Fig. 1A) with consensus sequence 5ЈTGTGNAT͞AAA (10, 11). Although R boxes have different affinities for DnaA based on slight differences in the nucleotide sequence, each R box binds inactive DnaA-ADP and active DnaA-ATP with equal affinity (8, 12). Based on this finding, two scenarios seem likely with regard to the requirement for DnaA-ATP in oriC unwinding. One possibility is that DnaA-ATP and Dna...

In an ongoing survey of the bioactive potential of microorganisms associated with marine invertebrates, the culture media of a sponge-associated bacterial strain of Pseudomonas aeruginosa was found to contain metabolites which inhibit the growth of several Gram-positive microorganisms. A series of diketopiperazines (1-6) including a new natural product (6) and two known phenazine alkaloid antibiotics (7 and 8) were isolated from the culture broth of this bacterium.

Summary 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 toward the center 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.

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.

DnaA occupies only the three highest-affinity binding sites in E. coli oriC throughout most of the cell cycle. Immediately prior to initiation of chromosome replication, DnaA interacts with additional recognition sites, resulting in localized DNA-strand separation. These two DnaA-oriC complexes formed during the cell cycle are functionally and temporally analogous to yeast ORC and pre-RC. After initiation, SeqA binds to hemimethylated oriC, sequestering oriC while levels of active DnaA are reduced, preventing reinitiation. In this paper, we investigate how resetting of oriC to the ORC-like complex is coordinated with SeqA-mediated sequestration. We report that oriC resets to ORC during sequestration. This was possible because SeqA blocked DnaA binding to hemimethylated oriC only at low-affinity recognition sites associated with GATC but did not interfere with occupation of higher-affinity sites. Thus, during the sequestration period, SeqA repressed pre-RC assembly while ensuring resetting of E. coli ORC.