The investigator is interested in understanding how bacterial plasmids are partitioned after cell division. He is using the plasmid pSC101 as a model system for studying the partition process. Previously, he has characterized a par locus on pSC101 that is essential for correct partitioning, and has identified some chromosomally encoded proteins (e.g., TopA, IHF) that can mitigate the effects of deleting par. He has evidence to suggest that the par locus does not encode a protein but rather acts in cis to alter plasmid DNA topology. Dr. Cohen's model is that RepA monomers bind to the origin region, together with DnaA and possibly other host proteins, to form a complex that is involved both in replication and partition (the P/R complex). It is this complex, not the par locus, that interacts with the cytoplasmic membrane during replication. The function of the par locus is to ensure the correct degree of supercoiling that promotes formation of this complex. The long term goal of the proposed work is to determine how RepA participates in replication and partitioning, and to determine how the partition/replication complex interacts with host proteins to mediate partitioning. Specifically, he proposes to (i) use ultracentrifugation , and ultimately X-ray crystallographic analysis of wild type and mutant RepAs (that compensate for lack of a par locus) to determine the relationship between RepA protein structure and its effects on partitioning and replication, (ii) identify and analyze host proteins that affect pSC101 partitioning at a step subsequent to formation of the partition-replication complex, (iii) use the two-hybrid system to identify host genes encoding proteins that interact with the RepA/DnaA/plasmid origin partition-replication complex, and (iv) investigate the temporal and physical relationship between partitioning of post-replicative plasmids, septation and cell division.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026355-16
Application #
2444490
Study Section
Special Emphasis Panel (ZRG5-BM-1 (03))
Project Start
1979-07-01
Project End
1999-06-30
Budget Start
1997-07-01
Budget End
1998-06-30
Support Year
16
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Stanford University
Department
Genetics
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
Miller, Christine; Ingmer, Hanne; Thomsen, Line Elnif et al. (2003) DpiA binding to the replication origin of Escherichia coli plasmids and chromosomes destabilizes plasmid inheritance and induces the bacterial SOS response. J Bacteriol 185:6025-31
Xu, Feng-Feng; Gaggero, Carina; Cohen, Stanley N (2002) Polyadenylation can regulate ColE1 type plasmid copy number independently of any effect on RNAI decay by decreasing the interaction of antisense RNAI with its RNAII target. Plasmid 48:49-58
Ingmer, H; Miller, C; Cohen, S N (2001) The RepA protein of plasmid pSC101 controls Escherichia coli cell division through the SOS response. Mol Microbiol 42:519-26
Miller, C; Cohen, S N (1999) Separate roles of Escherichia coli replication proteins in synthesis and partitioning of pSC101 plasmid DNA. J Bacteriol 181:7552-7
Ten Hagen, K G; Ravnan, J B; Cohen, S N (1995) Disparate replication properties of integrated and extrachromosomal forms of bovine papilloma virus in ID13 cells. J Mol Biol 254:119-29
Conley, D L; Cohen, S N (1995) Isolation and characterization of plasmid mutations that enable partitioning of pSC101 replicons lacking the partition (par) locus. J Bacteriol 177:1086-9
Conley, D L; Cohen, S N (1995) Effects of the pSC101 partition (par) locus on in vivo DNA supercoiling near the plasmid replication origin. Nucleic Acids Res 23:701-7
Ravnan, J B; Cohen, S N (1995) Transformed mouse cell lines that consist predominantly of cells maintaining bovine papilloma virus at high copy number. Virology 213:526-34
Ten Hagen, K G; Cohen, S N (1993) Timing of replication of beta satellite repeats of human chromosomes. Nucleic Acids Res 21:2139-42
Miller, C A; Cohen, S N (1993) The partition (par) locus of pSC101 is an enhancer of plasmid incompatibility. Mol Microbiol 9:695-702

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