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...

SummaryInitiator DnaA and DNA bending proteins, Fis and IHF, comprise prereplication complexes (pre-RC) that unwind the Escherichia coli chromosome's 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.

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.

SummaryPre-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 DnaAcatalysed 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 .

The RAZOR EX Anthrax Air Detection System, developed by Idaho Technology, Inc. (ITI), is a qualitative method for the detection of Bacillus anthracis spores collected by air collection devices. This system comprises a DNA extraction kit, a freeze-dried PCR reagent pouch, and the RAZOR EX real-time PCR instrument. Each pouch contains three assays, which distinguish potentially virulent B. anthracis from avirulent B. anthracis and other Bacillus species. These assays target the pXO1 and pXO2 plasmids and chromosomal DNA. When all targets are detected, the instrument makes an "anthrax detected" call, meaning that virulence genes of the anthrax bacillus are present. This report describes results from AOAC Method Developer (MD) and Independent Laboratory Validation (ILV) studies, which include matrix, inclusivity/exclusivity, environmental interference, upper and lower LOD of DNA, robustness, product consistency and stability, and instrument variation testing. In the MD studies, the system met the acceptance criteria for sensitivity and specificity, and the performance was consistent, stable, and robust for all components of the system. For the matrix study, the acceptance criteria of 95/96 expected calls was met for three of four matrixes, clean dry filters being the exception. Ninety-four of the 96 clean dry filter samples tested gave the expected calls. The nucleic acid limit of detection was 5-fold lower than AOAC's acceptable minimum detection limit. The system demonstrated no tendency for false positives when tested with Bacillus cereus. Environmental substances did not inhibit accurate detection of B. anthracis. The ILV studies yielded similar results for the matrix and inclusivity/exclusivity studies. The ILV environmental interference study included environmental substances and environmental organisms. Subsoil at a high concentration was found to negatively interfere with the pXO1 reaction. No interference was observed from the environmental organisms. The nucleic acid LOD, however, was 10 times higher (1 pg/reaction, equivalent to about 200 spores) than that found in the MD study. These results indicate that the RAZOR System is a sensitive and specific system that accurately identifies B. anthracis in aerosol matrixes and in the presence of interfering substances, and that the method can be performed by an independent laboratory and achieve similar results.