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

Resistance of tumors to drugs such as cisplatin and mitomycin C (MMC) is an important factor limiting their usefulness in cancer chemotherapy. The ant itumor effects of these drugs are due to the formation of bifunctional addu cts in DNA, with cisplatin causing predominantly intrastrand-crosslinks and MMC causing interstrand-crosslinks. The SOS chromotest was used to study t he cellular mechanisms that process DNA damage in Escherichia coli exposed to cisplatin, ultraviolet irradiation (UV) and MMC and subsequently facilit ate 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 (su lA) gene so that the amount of SOS inducing signal, which is modulated by t he ability of the cell to repair DNA, is measured by assaying beta -galacto sidase activity. SOS induction in a strain proficient in homologous recombi nation (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 pa thway caused by a recO mutation. The effect of cisplatin treatment in a uvr A 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 p ercent colony forming units (cfu) for cells treated with increasing doses o f cisplatin, MMC and UV relative to that in untreated control cultures. The importance of both HR pathways for resistance to these treatments was demo nstrated by decreased survival in mutants with the recB mutant being more s ensitive than the recO mutant. SOS induction levels were elevated in the se nsitive recB strain relative to the HR proficient strain possibly due to st alled and/or distorted replication forks at crosslinks in DNA. In contrast, induction of SOS was dependent on RecFOR activity that is thought to act a t daughter strand gaps in newly synthesized DNA to mediate production of th e signal for SOS induction. Proficiency in NER was necessary for both survi val and high levels of SOS induction in cisplatin treated cells. (C) 2001 P ublished by Elsevier Science B.V.