Abstract

Studies on plasmid replication have contributed significantly to our understanding of the regulation of DNA replication. Since bacterial plasmids are small DNA molecules tha replicate autonomously, they serve as useful systems for studies on the mechanism as well as regulation of DNA replication. Plasmids are intimately associated with antibiotic resistance and toxin production in a number of pathogens. Hence studies on plasmids are of considerable medical significance as well. We are investigating the mechanism and regulation of replication of a small Staphylococcus aureus plasmid, pT181. This plasmid is 4.4 kb in size and it encodes tetracycline resistance. We have isolated the pT181-encoded RepC protein that is required for its replication. We have also shown that RepC has a DNA relaxation activity that is specific for the pT181 origin. In addition, Repc creates a site-specific nick within the origin. The role of RepC protein in initiation, elongation and termination will be investigated by using anti-RepC antibodies to inhibit in vitro replication of pT181. Thermo-labile RepC protein will be isolated from strains carrying the plasmid pSA0301 and used for the determination of its role in termination, if any. By site-specific mutagenesis of the origin, nucleotides that are critical in replication will be identified. In vitro deletion analysis of the origin will be carried out to identify the minimal region that is required for replication and relaxation/nicking by RepC protein. In vitro replication experiments will be carried out in the presence of dideoxy NTPs, and the start-site of replication will be identified by 3' to 5' deletion analysis of the partially-replicated origin region using T4 DNA polymerase (in the absence of dNTPs). Mutants of RepC will be isolated by site-directed mutagenesis and studied for their DNA replication, relaxation/nicking and DNA binding properties. Amino acids in RepC that are critically associated with these activities will be identified. A radioimmunoassay using anti-RepC antibodies will be carried out to determine the amount of RepC in a number of pT181 copy mutants and to detect any direct relationship between the amount of RepC and the copy number.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031685-06
Application #
3279926
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1982-07-01
Project End
1990-08-31
Budget Start
1987-09-01
Budget End
1988-08-31
Support Year
6
Fiscal Year
1987
Total Cost
Indirect Cost

  Institution

Name
University of Pittsburgh
Department
Type
Schools of Medicine
DUNS #
053785812
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213

  Publications

Fagerburg, Matt V; Schauer, Grant D; Thickman, Karen R et al. (2012) PcrA-mediated disruption of RecA nucleoprotein filaments--essential role of the ATPase activity of RecA. Nucleic Acids Res 40:8416-24
Leuba, Sanford H; Anand, Syam P; Harp, Joel M et al. (2008) Expedient placement of two fluorescent dyes for investigating dynamic DNA protein interactions in real time. Chromosome Res 16:451-67
Anand, Syam P; Zheng, Haocheng; Bianco, Piero R et al. (2007) DNA helicase activity of PcrA is not required for the displacement of RecA protein from DNA or inhibition of RecA-mediated strand exchange. J Bacteriol 189:4502-9
Ruiz-Maso, J A; Anand, S P; Espinosa, M et al. (2006) Genetic and biochemical characterization of the Streptococcus pneumoniae PcrA helicase and its role in plasmid rolling circle replication. J Bacteriol 188:7416-25
Anand, Syam P; Chattopadhyay, Anasuya; Khan, Saleem A (2005) The PcrA3 mutant binds DNA and interacts with the RepC initiator protein of plasmid pT181 but is defective in its DNA helicase and unwinding activities. Plasmid 54:104-13
Khan, Saleem A (2005) Plasmid rolling-circle replication: highlights of two decades of research. Plasmid 53:126-36
Tinsley, Eowyn; Naqvi, Asma; Bourgogne, Agathe et al. (2004) Isolation of a minireplicon of the virulence plasmid pXO2 of Bacillus anthracis and characterization of the plasmid-encoded RepS replication protein. J Bacteriol 186:2717-23
Anand, Syam P; Mitra, Poulami; Naqvi, Asma et al. (2004) Bacillus anthracis and Bacillus cereus PcrA helicases can support DNA unwinding and in vitro rolling-circle replication of plasmid pT181 of Staphylococcus aureus. J Bacteriol 186:2195-9
Khan, Saleem A (2003) DNA-protein interactions during the initiation and termination of plasmid pT181 rolling-circle replication. Prog Nucleic Acid Res Mol Biol 75:113-37
Naqvi, Asma; Tinsley, Eowyn; Khan, Saleem A (2003) Purification and characterization of the PcrA helicase of Bacillus anthracis. J Bacteriol 185:6633-9

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