We propose to continue our work to investigate deeper into the mechanism of activation of a local origin gamma by initiator protein pi-ori interaction, activation of a distant ori by DNA looping, inactivation of an ori by handcuffing, in the plasmid R6K. We propose to use a newly reconstituted replication system consisting of 22 purified proteins to investigate the detailed mechanism of activation of the local origin gamma. We propose to identify the combination of chaperones (and any other proteins) needed to activate the dimeric and inert WT p so that it not only activates the local origin gamma, but is also able to activate the distant origins alpha and beta by DNA looping in vitro. The crystal structure of the monomeric initiator pi* bound to a single iteron DNA will be solved and the structure used to gain additional insights into the mechanism of initiation of replication at atomic resolution. Our long term goal is to extend crystallography further by co-crystallizing ternary complexes of peptides of DnaB, DnaA, DnaG that interact with p -iteron DNA complex with the objective of further extending our knowledge of replication initiation at high resolution.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI019881-23
Application #
6888901
Study Section
Special Emphasis Panel (ZRG1-CDF-2 (90))
Program Officer
Korpela, Jukka K
Project Start
1983-07-01
Project End
2009-04-30
Budget Start
2005-05-01
Budget End
2006-04-30
Support Year
23
Fiscal Year
2005
Total Cost
$458,877
Indirect Cost
Name
Medical University of South Carolina
Department
Biochemistry
Type
Schools of Medicine
DUNS #
183710748
City
Charleston
State
SC
Country
United States
Zip Code
29425
Vocke, C; Bastia, D (1985) The replication initiator protein of plasmid pSC101 is a transcriptional repressor of its own cistron. Proc Natl Acad Sci U S A 82:2252-6
Bastia, D; Vocke, C; Germino, J et al. (1985) DNA-protein interaction at the replication origins of plasmid chromosomes. Basic Life Sci 30:397-414
Kelley, W; Bastia, D (1985) Replication initiator protein of plasmid R6K autoregulates its own synthesis at the transcriptional step. Proc Natl Acad Sci U S A 82:2574-8