Structure-function aspects of control of DNA replication in prokaryotes

We are attempting to elucidate the mechanism(s) by which DNA synthesis is controlled in prokaryotes by studying structure-function relationships in isolated DNA-membrane complexes, the presumed site of replication in the prokaryotic cell.

Complexes have been isolated from B. subtilis which can initiate and direct DNA synthesis in vitro and which contain a variety of enzymes that catalyze synthesis of DNA precursors. Specific membrane-bound proteins that bind to the origin of replication have been isolated and are under investigation for their ability to effect DNA replication in vitro. One such membrane protein has been found to act as a repressor for initiation of DNA replication and may be related to a known respiratory enzyme. Plasmid derivatives derived from the broad host range plasmid RK2 cultured in E. coli have also been isolated in a membrane associated form. Such complexes carry out complete plasmid synthesis and may be bound to a specific subdomain of the E.coli inner membrane through its own initiation proteins. A region in the initiation protein containing a sequence of hydrophobic amino acids has been identified as the source of this interaction. This is the first demonstration of an initiation protein that has a domain for DNA binding and a domain for membrane binding. A novel gene related to the host DnaA initiation proteins (called the DnaA paralog) has been detected that physically interacts with the plasmid initiation proteins. Its protein product is half the size of the host initiation protein and may be involved in controlling the activity of the plasmid initiation proteins.

Recent Publications

Kim, P. D. Banack, T., Lerman, D., Tracy, J., Camara, J. E., Crooke, E., Oliver, D., and Firshein, W. (2003). Identification of a novel membrane associated gene product that suppresses the toxicity of a TrfA peptide from plasmid RK2 and its relations to the DnaA host initiation protein. J. Bacteriology 185:1817-1824.

Firshein, W. (2002) Prokaryotic DNA Replication: In: Modern Microbial Genetics, 2nd Edition. Ed: UN Streips and R. E. Yasbin, Wiley-Liss; pp. 3-27.

Firshein, W. (2002) Review of Horizontal Gene Transfer, 2nd Ed., Ed. M. Syvanen and C. I. Kado. Plasmid 48:229-231.

Kim, P. D., Rosche, T., and Firshein, W. (2000). Identification of a potential membrane targeting region of the replication initiation protein (TrfA) of broad host range plasmid RK2. Plasmid 43:214-222.

Kim, P. D. and Firshein W. (2000) Isolation of an inner membrane derived subfraction that supports in vitro replication of a mini RK2 plasmid in Escherichia coli. J. Bacteriol. 182:1757-1760.

Stein, A., and Firshein, W. (2000). Probable identification of a membrane associated repressor of Bacillus subtilis DNA replication as the E2 subunit of the pyruvate dehydrogenase complex. J. Bacteriol. 182:2119-2124.

Banack, T., Kim, P.D., and Firshein, W. (2000). TrfA dependent inner membrane associated plasmid RK2 DNA synthesis and the association of this protein with membranes of different Gram Negative hosts. J. Bacteriol. 182:4380-4383.

Firshein, W and Kim, P. (1997). Plasmid replication and partition in E. coli: Is the cell membrane the key? Mol. Microbiol. 23:(1) 1-10.