Escherichia coli DnaA protein binds to the chromosomal origin to initiate chromosomal DNA replication. In contrast, induced expression of dnaA+ from a multicopy plasmid increases initiation frequency but viability is maintained.
Further work on how cells maintain the integrity of its genome may lead to advances in agriculture and medicine.Publications, Progress 01/01/11 to 12/31/11OutputsOUTPUTS: The long-term goals of this project are to understand the mechanism of initiation of chromosomal DNA replication and its regulation in E. coli. Also, we showed that DnaA interacts specifically with HU protein composed of two alpha subunits. We studied a dnaA mutation named dnaAcos, which is lethal because it induces extra initiations. This project investigates the molecular mechanisms of initiation of DNA replication and its regulation.
Published Despite their ability to deliver DnaB to the chromosomal origin to form the prepriming complex, this intermediate is inactive.
We suggest that the presence of this gene in E. coli provides a growth advantage.
26: 5170-5175.
19 pp. The E. coli dnaA gene: four functional domains. The involvement of DnaK protein in replication described in the previous progress report, and observations of others that expression of dnaK in Caulobacter crescentus occurs at a specific time in the cell cycle concomitant with After isolating a small collection of novel dnaA mutations, we showed that the mutant DnaAs promote excessive initiations, and suggest that they presumably fail to respond to regulatory factors that normally control the frequency of initiation. 2014
1997.
By lowering the steady-state level of double strand breaks, it helps to maintain the integrity of the bacterial chromosome. The helicase then acts at the newly formed replication forks to unwind the parental duplex in advance of DNA replication. 274: 546-561. Chem. Year Published: Results obtained during this funding period provide insight into these processes.
This set of experiments tests the hypothesis that DnaC acts as a molecular switch that controls whether DnaB can interact with primase or not. (2011) Understanding protein function, the disparity between bioinformatics and molecular methods.
By focussing on the role of the dnaA gene product, the long range objectives of this research are to understand biochemically the initiation of chromosomal replication, and its regulation. Domains of DnaA protein involved in DNA binding, and ATP binding have been identified. Ludlam, A. V., McNatt, M. Carr, K. Kaguni, J.M.
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DnaC binds to DnaB to form the DnaB6-DnaC6 complex. Hupert-Kocurek, K., Sage, J. M., Makowska-Grzyska, M., and Kaguni, J. M. 2007.
Molecular Mechanisms in DNA Replication and Recombination. Citation: MARSZALEK, J., and KAGUNI, J.M.
* During replication, the two strands separate with one strand serving as the template.
Only four inhibit DNA replication. Lane, eds.
273: 24355-34262. 2002. Progress in the last granting period includes purification of a factor and its identification as grpE protein.
The results indicate that all forms of HU were equally active. A mutant form, DnaAcos carrying four amino acid substitutions, is apparently defective in sensing regulatory signals because it induces hyperactive initiation.
This intermediate is formed on a single stranded DNA that is duplicated via a process that is very similar to that which occurs at the E. coli chromosomal origin. Those that bind ATP but fail to hydrolyze it are lethal and will not be obtained. DnaC forms a complex with DnaB helicase, and this complex is required for this helicase to bind to the E. coli chromosomal origin. (2011) DNA Replication: Initiation in Bacteria. This event starts by the step-wise assembly of a nucleoprotein complex termed the replisome at specific sites in the genome called origins of replication.
We have recently constructed expression vectors in which wild type or mutant dnaA genes have been placed under T7 promoter control. Based on suppression of the lethal effect caused by elevated dnaA expression, we developed a genetic method to select for multicopy suppressors that regulate the frequency of initiation, and obtained plasmids carrying seqA, datA, dnaN and hda, which respectively are known either to act at the replication origin (oriC), or to modulate the activity of DnaA protein. 1442: 39-48. Conference Papers and Presentations
One monoclonal antibody that interferes with the interaction between DnaA protein and DnaB helicase implicates a region of DnaA protein involved in this interaction. Escherichia coli DnaA protein: Specific biochemical defects of mutant DnaAs reduce initiation frequency to suppress a temperature-sensitive dnaX mutation. DnaA protein functions at initiation to regulate the frequency of initiation. By use of a synchrony method, the rate of synthesis of DnaK protein did not change dramatically relative to the cell cycle (manuscript in preparation). Drs.
177, 6657-6665. Book Chapters
The important observation is that the number of DnaB molecules in the prepriming complex is restricted, suggesting a mechanism to control this event.ImpactsChromosomal DNA replication is essential in all free-living organisms. 1998.
Elsevier Science. Escherichia coli DnaA interacts with HU in initiation at the E. coli replication origin: E. coli HU protein is a dimer encoded by two closely related genes whose expression is growth-phase dependent. These double strand breaks are toxic in mutants that are unable to repair them. Because DNA replication is regulated during the initiation process, this protein has been the focus of study with long range objectives of understanding biochemically the initiation of chromosomal replication, and its regulation.
To test this model, we used a replication system with a single-stranded DNA template carrying a DnaA box sequence in a hairpin structure. On entry of DnaB at the E. coli chromosomal origin,oriC, ATP hydrolysis releases DnaC, a step that is essential to unmask the helicase activity of DnaB. Finally, biochemical characterization of DnaA protein indicates that it binds in an ordered manner to the chromosomal origin.Impacts(N/A)Publications, Progress 01/01/93 to 12/30/93OutputsThe long range objectives of this research are to understand biochemically the initiation of chromosomal replication, and its regulation. 1998. In conjunction, we have mapped epitopes recognized by a collection of monoclonal The interaction of primase with DnaB, and the release of DnaC mark discrete events in the transition from initiation to the elongation stage of DNA replication.Publications, Progress 01/01/09 to 12/31/09OutputsOUTPUTS: DnaAcos hyperinitiates by circumventing regulatory pathways that control the frequency of initiation in Escherichia coli - Previously, we showed that elevated levels of DnaA protein induce extra initiations by apparently overcoming regulatory pathways that control the frequency of initiation of DNA replication in E. coli.
In other work, we studied specific dnaA alleles that reduce initiation frequency and chromosome content per cell at 34 to 39oC and fail to support initiation at both 20 and 44oC.
Escherichia coli DnaA Protein: The replication initiator. In E. coli, chromosomal replication occurs at a specific time in the bacterial cell cycle and is a highly regulated event. If you are on a personal connection, like at home, you can run an anti-virus scan on your device to make sure it is not infected with malware. Environ.