Blocking, Bending, and Binding: Regulation of Initiation of Chromosome Replication During the Cell Cycle by Transcriptional Modulators That Interact With Origin DNA.

Genome duplication is a critical event in the reproduction cycle of every cell. Because all daughter cells must inherit a complete genome, chromosome replication is tightly regulated, with multiple mechanisms focused on controlling when chromosome replication begins during the cell cycle. In bacteria, chromosome duplication starts when nucleoprotein complexes, termed orisomes, unwind replication origin () DNA and recruit proteins needed to build new replication forks.

Changing Perspectives on the Role of DnaA-ATP in Orisome Function and Timing Regulation.

Bacteria, like all cells, must precisely duplicate their genomes before they divide. Regulation of this critical process focuses on forming a pre-replicative nucleoprotein complex, termed the orisome. Orisomes perform two essential mechanical tasks that configure the unique chromosomal replication origin, to start a new round of chromosome replication: (1) unwinding origin DNA and (2) assisting with loading of the replicative DNA helicase on exposed single strands.

Blocking the Trigger: Inhibition of the Initiation of Bacterial Chromosome Replication as an Antimicrobial Strategy.

All bacterial cells must duplicate their genomes prior to dividing into two identical daughter cells. Chromosome replication is triggered when a nucleoprotein complex, termed the orisome, assembles, unwinds the duplex DNA, and recruits the proteins required to establish new replication forks. Obviously, the initiation of chromosome replication is essential to bacterial reproduction, but this process is not inhibited by any of the currently-used antimicrobial agents.

Low Affinity DnaA-ATP Recognition Sites in Make Non-equivalent and Growth Rate-Dependent Contributions to the Regulated Timing of Chromosome Replication.

Although the mechanisms that precisely time initiation of chromosome replication in bacteria remain unclear, most clock models are based on accumulation of the active initiator protein, DnaA-ATP. During each cell division cycle, sufficient DnaA-ATP must become available to interact with a distinct set of low affinity recognition sites in the unique chromosomal replication origin, , and assemble the pre-replicative complex (orisome) that unwinds origin DNA and helps load the replicative helicase. The low affinity -DnaA-ATP interactions are required for the orisome's mechanical functions, and may also play a role in timing of new rounds of DNA synthesis.

Origin recognition is the predominant role for DnaA-ATP in initiation of chromosome replication.

In all cells, initiation of chromosome replication depends on the activity of AAA+ initiator proteins that form complexes with replication origin DNA. In bacteria, the conserved, adenosine triphosphate (ATP)-regulated initiator protein, DnaA, forms a complex with the origin, oriC, that mediates DNA strand separation and recruitment of replication machinery. Complex assembly and origin activation requires DnaA-ATP, which differs from DnaA-ADP in its ability to cooperatively bind specific low affinity sites and also to oligomerize into helical filaments.

Targeting the Bacterial Orisome in the Search for New Antibiotics.

There is an urgent need for new antibiotics to combat drug resistant bacteria. Existing antibiotics act on only a small number of proteins and pathways in bacterial cells, and it seems logical that expansion of the target set could lead to development of novel antimicrobial agents. One essential process, not yet exploited for antibiotic discovery, is the initiation stage of chromosome replication, mediated by the bacterial orisome.

Nucleotide-Induced Conformational Changes in Escherichia coli DnaA Protein Are Required for Bacterial ORC to Pre-RC Conversion at the Chromosomal Origin.

DnaA oligomerizes when bound to origins of chromosomal replication. Structural analysis of a truncated form of DnaA from Aquifex aeolicus has provided insight into crucial conformational differences within the AAA+ domain that are specific to the ATP- versus ADP- bound form of DnaA. In this study molecular docking of ATP and ADP onto Escherichia coli DnaA, modeled on the crystal structure of Aquifex aeolicus DnaA, reveals changes in the orientation of amino acid residues within or near the vicinity of the nucleotide-binding pocket.

The orisome: structure and function.

During the cell division cycle of all bacteria, DNA-protein complexes termed orisomes trigger the onset of chromosome duplication. Orisome assembly is both staged and stringently regulated to ensure that DNA synthesis begins at a precise time and only once at each origin per cycle. Orisomes comprise multiple copies of the initiator protein DnaA, which oligomerizes after interacting with specifically positioned recognition sites in the unique chromosomal replication origin, oriC.

Building the bacterial orisome: high-affinity DnaA recognition plays a role in setting the conformation of oriC DNA.

During assembly of the E. coli pre-replicative complex (pre-RC), initiator DnaA oligomers are nucleated from three widely separated high-affinity DnaA recognition sites in oriC. Oligomer assembly is then guided by low-affinity DnaA recognition sites, but is also regulated by a switch-like conformational change in oriC mediated by sequential binding of two DNA bending proteins, Fis and IHF, serving as inhibitor and activator respectively.

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.

Remodeling of nucleoprotein complexes is independent of the nucleotide state of a mutant AAA+ protein.

DnaA protein, a member of the AAA+ (ATPase associated with various cellular activities) family, initiates DNA synthesis at the chromosomal origin of replication (oriC) and regulates the transcription of several genes, including its own. The assembly of DnaA complexes at chromosomal recognition sequences is affected by the tight binding of ATP or ADP by DnaA. DnaA with a point mutation in its membrane-binding amphipathic helix, DnaA(L366K), previously described for its ability to support growth in cells with altered phospholipid content, has biochemical characteristics similar to those of the wild-type protein.

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.

Regulating DnaA complex assembly: it is time to fill the gaps.

New rounds of bacterial chromosome replication are triggered during each cell division cycle by the initiator protein, DnaA. For precise timing, interactions of DnaA-ATP monomers with the replication origin, oriC, must be carefully regulated during formation of complexes that unwind origin DNA and load replicative helicase. Recent studies in Escherichia coli suggest that high and low affinity DnaA recognition sites are positioned within oriC to direct staged assembly of bacterial pre-replication complexes, with DnaA contacting low affinity sites as it oligomerizes to 'fill the gaps' between high affinity sites.

Initiation of DNA Replication.

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.

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.

Initiating chromosome replication in E. coli: it makes sense to recycle.

Initiating new rounds of Escherichia coli chromosome replication requires DnaA-ATP to unwind the replication origin, oriC, and load DNA helicase. In this issue of Genes & Development, Fujimitsu and colleagues (pp. 1221-1233) demonstrate that two chromosomal sites, termed DARS (DnaA-reactivating sequences), recycle inactive DnaA-ADP into DnaA-ATP.

Fetal cardiac diagnosis and its influence on the pregnancy and newborn--a tertiary centre experience.

Objectives: To evaluate the impact of an abnormal fetal cardiac scan on the management of the pregnancy and the outcome of the newborn.

Methods: We reviewed all pregnancies that were referred to the Fetal Cardiac Unit for assessment to determine if the finding of a cardiac abnormality influenced the pregnancy and fetus, timing and mode of delivery, the treatment and outcome of the newborn. Diagnoses were confirmed by echocardiography following the baby's delivery.

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

Prior to initiating DNA synthesis, Escherichia coli oriC switches from ORC, comprising initiator DnaA bound at three high-affinity sites, to pre-RC, when additional DnaA molecules interact with low-affinity sites. Two types of low-affinity sites exist: R boxes that bind DnaA-ATP and DnaA-ADP with equal affinity, and I-sites with a three- to fourfold preference for DnaA-ATP. To assess the regulatory role of weak DnaA interactions during pre-RC assembly in vivo, we compared the behaviour of plasmid-borne wild-type oriC with mutants having an increased or decreased number of DnaA-ATP discriminatory I-sites.

SeqA blocking of DnaA-oriC interactions ensures staged assembly of the E. coli pre-RC.

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.

The intrinsic ATPase activity of Mycobacterium tuberculosis DnaA promotes rapid oligomerization of DnaA on oriC.

Oligomerization of the initiator protein, DnaA, on the origin of replication (oriC) is crucial for initiation of DNA replication. Studies in Escherichia coli (Gram-negative) have revealed that binding of DnaA to ATP, but not hydrolysis of ATP, is sufficient to promote DnaA binding, oligomerization and DNA strand separation. To begin understanding the initial events involved in the initiation of DNA replication in Mycobacterium tuberculosis (Gram-positive), we investigated interactions of M.

Counselling strategies in the prenatal diagnosis of major heart abnormality.

Background: Prenatal diagnosis of a major cardiac abnormality precipitates a crisis in the parents. To their grief and/or anger may be added the burden of whether to continue with the pregnancy if the fetus is previable.

Aims: We reviewed our counselling strategies to determine if the parents were satisfied with our approach and whether in their perception it provided the knowledge and understanding of the abnormality detected to help them in their decision.

Pre-natal counselling--helping couples make decisions following the diagnosis of severe heart disease.

The prenatal diagnosis of a major cardiac abnormality tends to precipitate a crisis for the affected parents. In a setting of grief and emotional distress, there is the challenge to provide meaningful information of the abnormality, its need for intervention and likely outcome, so as to enable the parents, if allowed the option, to come to a fully informed decision as to whether to continue with the pregnancy. This discussion paper reviews the difficulties encountered in counselling affected parents being mindful of the psychological constraints prevalent at the time.

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.

Effective counselling of pre-natal diagnosis of serious heart disease--an aid to maternal bonding?

Unlabelled: The pre-natal diagnosis of a serious anomaly in the fetus precipitates a crisis for the parents. Effective counselling may help during this difficult phase. We postulated that if parents opted to continue with the pregnancy, then they are better prepared to care for the newborn infant having already experienced the grieving process.

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 chromosome's origin of replication, oriC. Loss of either Fis or IHF perturbs synchronous initiation from oriC copies in rapidly growing E. coli.