title: Survival and induction of SOS in Escherichia coli treated with cisplatin, UV-irradiation, or mitomycin C are dependent on the function of the RecBC and RecFOR pathways of homologous recombination.
abstract: Resistance of tumors to drugs such as cisplatin and mitomycin C (MMC) is an important factor limiting their usefulness in cancer chemotherapy. The antitumor effects of these drugs are due to the formation of bifunctional adducts in DNA, with cisplatin causing predominantly intrastrand-crosslinks and MMC causing interstrand-crosslinks. The SOS chromotest was used to study the cellular mechanisms that process DNA damage in Escherichia coli exposed to cisplatin, ultraviolet irradiation (UV) and MMC and subsequently facilitate the production of a molecular signal for induction of the SOS response. Strains used in the SOS chromotest have a fusion of lacZ with the sfiA (sulA) gene so that the amount of SOS inducing signal, which is modulated by the ability of the cell to repair DNA, is measured by assaying beta-galactosidase activity. SOS induction in a strain proficient in homologous recombination (HR) was compared with that in isogenic strains deficient in HR due to a blocked RecBC pathway caused by a recB mutation or a blocked RecFOR pathway caused by a recO mutation. The effect of cisplatin treatment in a uvrA mutant strain blocked at the first step of NER was compared with that in an isogenic strain proficient in NER. Cellular resistance was measured as percent colony forming units (cfu) for cells treated with increasing doses of cisplatin, MMC and UV relative to that in untreated control cultures. The importance of both HR pathways for resistance to these treatments was demonstrated by decreased survival in mutants with the recB mutant being more sensitive than the recO mutant. SOS induction levels were elevated in the sensitive recB strain relative to the HR proficient strain possibly due to stalled and/or distorted replication forks at crosslinks in DNA. In contrast, induction of SOS was dependent on RecFOR activity that is thought to act at daughter strand gaps in newly synthesized DNA to mediate production of the signal for SOS induction. Proficiency in NER was necessary for both survival and high levels of SOS induction in cisplatin treated cells.
authors.affiliation: Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403, USA.
authors.forename: K L
authors.initials: KL
authors.lastname: Keller
authors.forename: T L
authors.initials: TL
authors.lastname: Overbeck-Carrick
authors.forename: D J
authors.initials: DJ
authors.lastname: Beck
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D002945:Cisplatin / Q000494:pharmacology*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D004260:DNA Repair / Q000187:drug effects / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004351:Drug Resistance / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005838:Genotype; D016685:Mitomycin / Q000494:pharmacology*; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010641:Phenotype; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D011995:Recombination, Genetic; D013014:SOS Response, Genetics; D015398:Signal Transduction; D014466:Ultraviolet Rays
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D011838:Radiation-Sensitizing Agents; C081006:RecO protein, E coli; C489318:RecR protein, E coli; C489340:recF protein, E coli; C065831:RecR protein, Bacteria; D016685:Mitomycin; C066218:recF protein, Bacteria; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D002945:Cisplatin