This proposal focuses on formation of DNA-protein complexes that trigger the onset of chromosome duplication, with the long term objective of understanding molecular mechanisms that control bacterial cell growth. In bacteria, the rate of initiation of chromosome replication determines the frequency of cell duplication and requires assembly of a pre-replication complex (pre-RC), comprising multiple copies of the conserved initiator DnaA bound to an origin of replication, oriC. Our hypothesis is that the oriC sequence contains all the information required to ensure that the pre-RC is assembled at the correct time in the cell cycle at all growth rates. We base this hypothesis on our observations that: 1) E. coli oriC contains high affinity DnaA binding sites that are occupied throughout the cell cycle, forming a bacterial Origin Recognition Complex (ORC);2) The ORC sites flank arrays of lower affinity sites which interact with DnaA to form the pre-RC only at the time of initiation of chromosome replication;thus occupancy of low affinity sites determines initiation timing;3) Low affinity sites are targets for multiple mechanisms that regulate initiation by modulating DnaA binding;and 4) DnaA cannot occupy low affinity sites in the absence of ORC. Based on these observations our goals for this proposal are to define how arrangement of high and low affinity DnaA binding sites in E. coli oriC directs ORC to pre-RC transition and regulates timing of initiation during the cell cycle, and to understand how this transition may be regulated by growth rate-dependent mechanisms.
The Specific Aims are to: 1) Test the hypothesis that loading and stable occupation of low affinity DnaA recognition sites is dependent on proximal ORC sites;2) Test the hypothesis that loading of phased arrays of low affinity DnaA recognition sites is a late stage of pre-RC formation that determines the requirement for DnaA-ATP and regulates initiation timing during the cell cycle;and 3) Test the hypothesis that growth rate regulated binding of Fis and IHF to oriC modulates the level of DnaA required for low affinity site occupation. Accomplishing these aims will advance our understanding of how sequence information in bacterial origins directs precisely timed, cell cycle specific complex assembly. This information will improve our understanding of growth regulation in both enteric and non-enteric pathogens. Studies described here will also aid in the identification of new targets appropriate to guide the design of novel cell growth inhibitors for bacterial pathogens.
The long term goal of the proposed research is to understand mechanisms that control cell growth. Understanding these mechanisms will be useful in identifying new targets to guide the design of cell growth inhibitors to treat cancer or diseases caused by pathogenic bacteria.
|Kaur, Gulpreet; Vora, Mansi P; Czerwonka, Christopher A et al. (2014) Building the bacterial orisome: high-affinity DnaA recognition plays a role in setting the conformation of oriC DNA. Mol Microbiol 91:1148-63|
|Leonard, Alan C; Mechali, Marcel (2013) DNA replication origins. Cold Spring Harb Perspect Biol 5:a010116|
|Leonard, Alan C; Grimwade, Julia E (2011) Regulation of DnaA assembly and activity: taking directions from the genome. Annu Rev Microbiol 65:19-35|
|Saxena, Rahul; Rozgaja, Tania; Grimwade, Julia et al. (2011) Remodeling of nucleoprotein complexes is independent of the nucleotide state of a mutant AAA+ protein. J Biol Chem 286:33770-7|
|Rozgaja, Tania A; Grimwade, Julia E; Iqbal, Maryam et al. (2011) Two oppositely oriented arrays of low-affinity recognition sites in oriC guide progressive binding of DnaA during Escherichia coli pre-RC assembly. Mol Microbiol 82:475-88|
|Miller, Diana T; Grimwade, Julia E; Betteridge, Thu et al. (2009) Bacterial origin recognition complexes direct assembly of higher-order DnaA oligomeric structures. Proc Natl Acad Sci U S A 106:18479-84|
|Leonard, Alan C; Grimwade, Julia E (2009) Initiating chromosome replication in E. coli: it makes sense to recycle. Genes Dev 23:1145-50|
|Grimwade, Julia E; Torgue, Julien J-C; McGarry, Kevin C et al. (2007) Mutational analysis reveals Escherichia coli oriC interacts with both DnaA-ATP and DnaA-ADP during pre-RC assembly. Mol Microbiol 66:428-39|
|Madiraju, Murty V V S; Moomey, Meredith; Neuenschwander, Pierre F et al. (2006) The intrinsic ATPase activity of Mycobacterium tuberculosis DnaA promotes rapid oligomerization of DnaA on oriC. Mol Microbiol 59:1876-90|
|Nievera, Christian; Torgue, Julien J-C; Grimwade, Julia E et al. (2006) SeqA blocking of DnaA-oriC interactions ensures staged assembly of the E. coli pre-RC. Mol Cell 24:581-92|
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