Presentation Type
Poster
Start Date
5-2-2018 11:00 AM
End Date
5-2-2018 1:00 PM
Subjects
Escherichia coli -- Genetics -- Experiments, Photochemotherapy, DNA repair, Genetic recombination, Psoralens
Abstract
8-methoxypsoralen is a photoactivated DNA-intercalating agent, which after absorbing two high-energy UVA photons, covalently binds pyrimidine bases on both DNA strands, to form an interstrand crosslink (ICL). These lesions completely block replication and transcription, and are widely used in chemotherapies; yet the mechanism by which they are processed remains poorly understood. In 1985, Ahmed and Holland reported an Escherichia coli mutant demonstrating hyper-resistance to ICL-inducing agents. The mutation was mapped to 57.2 minutes on the chromosome, and potentially encoded a 55-kDa protein that was induced as part of the SOS response. Although these genes remain unidentified, hscA and hscB map to this location, have a similar size, and are SOS-inducible. To determine if these genes or others might confer ICL resistance in E. coli, we characterized how cells survived psoralen-UVA (PUVA) treatment in the absence of HscAB, and when these gene products were overexpressed. In a second approach to screen for ICL resistance genes, we developed a selection system to isolate hyper-resistant strains through the sequential growth and exposure of wild-type cultures to PUVA. We found no effect on cell survival in the hscAB mutant compared to its wild-type parent, suggesting that HscAB may not be contributing to ICL resistance as previously hypothesized. However, due to the significant cytotoxicity of plasmids containing hscAB, even in the absence of PUVA treatment, we were unable to determine whether over-expression of these gene products might provide a protective effect to cells. Using iteratively PUVA cells, we isolated strains that were >10^4-fold more resistant to this ICL-inducing agent compared to the parent strain. This result suggests that E. coli possess mechanisms to repair or tolerate ICL’s that contribute to resistance to these agents, similar to what is observed in human cancer cells.
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Persistent Identifier
http://archives.pdx.edu/ds/psu/25030
Included in
Interstrand Crosslink Resistance in Escherichia Coli
8-methoxypsoralen is a photoactivated DNA-intercalating agent, which after absorbing two high-energy UVA photons, covalently binds pyrimidine bases on both DNA strands, to form an interstrand crosslink (ICL). These lesions completely block replication and transcription, and are widely used in chemotherapies; yet the mechanism by which they are processed remains poorly understood. In 1985, Ahmed and Holland reported an Escherichia coli mutant demonstrating hyper-resistance to ICL-inducing agents. The mutation was mapped to 57.2 minutes on the chromosome, and potentially encoded a 55-kDa protein that was induced as part of the SOS response. Although these genes remain unidentified, hscA and hscB map to this location, have a similar size, and are SOS-inducible. To determine if these genes or others might confer ICL resistance in E. coli, we characterized how cells survived psoralen-UVA (PUVA) treatment in the absence of HscAB, and when these gene products were overexpressed. In a second approach to screen for ICL resistance genes, we developed a selection system to isolate hyper-resistant strains through the sequential growth and exposure of wild-type cultures to PUVA. We found no effect on cell survival in the hscAB mutant compared to its wild-type parent, suggesting that HscAB may not be contributing to ICL resistance as previously hypothesized. However, due to the significant cytotoxicity of plasmids containing hscAB, even in the absence of PUVA treatment, we were unable to determine whether over-expression of these gene products might provide a protective effect to cells. Using iteratively PUVA cells, we isolated strains that were >10^4-fold more resistant to this ICL-inducing agent compared to the parent strain. This result suggests that E. coli possess mechanisms to repair or tolerate ICL’s that contribute to resistance to these agents, similar to what is observed in human cancer cells.