cisplatin

RecN and RecG are Required for Bleomycin Survival by Escherichia coli

Reference: Kosa, J.L., Zdraveski, Z.Z., Currier, S., Marinus, M.G. and Essigmann, J.M. 2004. RecN and RecG are Required for Bleomycin Survival by Escherichia coli. Mutation Resaerch 554, 149-157.

The sensitivity of a panel of DNA repair-defective bacterial strains to BLM was investigated. E. coli recA cells were far more sensitive than were uvrA, dam-3, and mutM mutY strains, underscoring the importance of RecA to survival. Strains recBCD and recN, which lack proteins required for double strand break (DSB) repair, were extremely sensitive to BLM, while recF cells were not. The requirement for DSB-specific enzymes supports the hypothesis that DSBs are the primary cause of bleomycin cytotoxicity. The extreme sensitivity of recN cells was comparable to that of recA, implying a central role for the RecN protein in BLM lesion repair. The Holliday junction processing enzymes RecG and RuvC were both required for BLM survival. The recG ruvC double mutant was no more sensitive than either mutation alone, suggesting that both enzymes participate in the same pathway. Surprisingly, ruvAB cells were no more sensitive than wildtype, implying that RuvC is able to perform its role without RuvAB. This observation contrasts with current models of recombination in which RuvA,B, and C function as a single complex. The most straightforward explanation of these results is that DSB repair involves a structure that serves as a good substrate for RecG, and not RuvAB.

Reference: Zdravesky, Z.Z., Mello, J.A., Marinus, M.G. and Essigmann, J.M. 2000. Multiple pathways of recombination define cellular responses to cisplatin. Chemistry & Biology 7, 39-50.

Zdraveski ZZ, Mello JA, Farinelli CK, Essigmann JM, Marinus MG. (2002) MutS preferentially recognizes cisplatin- over oxaliplatin-modified DNA. J Biol Chem 277, 1255-60.

Loss of mismatch repair leads to tumor resistance by desensitizing cells to specific DNA-damaging agents, including the anticancer drug cisplatin. Cisplatin analogs with a diamminocyclohexane (DACH) carrier ligand, such as oxaliplatin and Pt(DACH)Cl(2), do not elicit resistance in mismatch repair-deficient cells and therefore present promising therapeutic agents. This study compared the interactions of the purified Escherichia coli mismatch repair protein MutS with DNA modified to contain cisplatin and DACH adducts. MutS recognized the cisplatin-modified DNA with 2-fold higher affinity in comparison to the DACH-modified DNA. ADP stimulated the binding of MutS to cisplatin-modified DNA, whereas it had no effect on the MutS interaction with DNA modified by DACH or EN adducts. In parallel cytotoxicity experiments, methylation-deficient E. coli dam mutants were 2-fold more sensitive to cisplatin than DACH compounds. A panel of recombination-deficient mutants showed striking sensitivity to both compounds, indicating that both types of adducts are strong replication blocks. The differential affinity of MutS for DNA modified with the different platinum analogs could provide the molecular basis for the distinctive cellular responses to cisplatin and oxaliplatin.

Reference: Spek, E.J., Wright, T.I., Stitt, M.S., Taghizadeh, N.R., Tannenbaum, S.R., Marinus, M.G.and Engelward, B.P. (2001). Recombinational repair is critical for the survival of Escherichia coli exposed to nitric oxide. J. Bacteriol. 183, 131-138.

Nitric Oxide (NO.) is critical to numerous biological processes, including signal transduction and macrophage mediated immunity. In this study, we have explored the biological effects of NO. induced DNA damage in Escherichia coli. The relative importance of base excision repair (BER), nucleotide excision repair (NER) and recombinational repair in preventing NO. induced toxicity was determined. E. colialkA tag], oxidative damage [fpg nei nth], and deaminated cytosine [ung]) showed essentially wild type levels of NO. resistance. However, AP endonuclease deficient cells (xth nfo) were very sensitive to killing by NO., which indicates that normal processing of AP sites is critical for defending against NO.. In addition, recA mutant cells are exquisitely sensitive to NO. induced killing. Both SOS deficient (lexA3) and Holliday junction resolvase deficient (ruvC) cells are very sensitive to NO., indicating that both SOS and recombinational repair play very important roles in defending against NO.. Furthermore, strains specifically lacking double strand end repair (recBCD) are very sensitive to NO., and such cells accumulate NO. induced double strand ends (as shown by pulse field gel electrophoresis). One consequence of these double strand ends is that NO. induces recombination at a genetically engineered substrate. Taken together, these results point to recombinational repair as a potential susceptibility factor for invading microbes and also suggest that it is important to account for both the potential of NO. to induce point mutations and recombination events when considering the effects of NO. exposure lacking either NER or DNA glycosylases (including those that repair alkylation damage.

Reference: Spek EJ, Vuong LN, Matsuguchi T, Marinus MG, Engelward BP.2002. Nitric Oxide-Induced Homologous Recombination in Escherichia coli Is Promoted by DNA Glycosylases. J Bacteriology 184, 3501-3507

Nitric oxide (NO(.)) is involved in neurotransmission, inflammation, and many other biological processes. Exposure of cells to NO(.) leads to DNA damage, including formation of deaminated and oxidized bases. Apurinic/apyrimidinic (AP) endonuclease-deficient cells are sensitive to NO(.) toxicity, which indicates that base excision repair (BER) intermediates are being generated. Here, we show that AP endonuclease-deficient cells can be protected from NO(.) toxicity by inactivation of the uracil (Ung) or formamidopyrimidine (Fpg) DNA glycosylases but not by inactivation of a 3-methyladenine (AlkA) DNA glycosylase. These results suggest that Ung and Fpg remove nontoxic NO(.)-induced base damage to create BER intermediates that are toxic if they are not processed by AP endonucleases. Our next goal was to learn how Ung and Fpg affect susceptibility to homologous recombination. The RecBCD complex is critical for repair of double-strand breaks via homologous recombination. When both Ung and Fpg were inactivated in recBCD cells, survival was significantly enhanced. We infer that both Ung and Fpg create substrates for recombinational repair, which is consistent with the observation that disrupting ung and fpg suppressed NO(.)-induced recombination. Taken together, a picture emerges in which the action of DNA glycosylases on NO(.)-induced base damage results in the accumulation of BER intermediates, which in turn can induce homologous recombination. These studies shed light on the underlying mechanism of NO(.)-induced homologous recombination.

Reference: Nowosielska, A., Calmann, M.A., Zdraveski, Z., Essigmann, J.M. and Marinus, M.G. (2004) Spontaneous and cisplatin-induced recombination in Escherichia coli. DNA Repair 3, 719-728.

To measure cisplatin-induced recombination, we have used a qualitative intrachromosomal assay utilizing duplicate inactive lac operons containing non-overlapping deletions and selection for Lac⁺ recombinants. The two operons are separated by one Mb and conversion of one of them yields the Lac⁺ phenotype. Lac⁺ formation for both spontaneous and cisplatin-induced recombination requires the products of the recA, recBC, ruvA, ruvB, ruvC, priA and polA genes. Inactivation of the recF, recO, recR and recJ genes decreased cisplatin-induced, but not spontaneous, recombination. The dependence on PriA and RecBC suggests that recombination is induced following stalling or collapse of replication forks at DNA lesions to form double strand breaks. The lack of recombination induction by trans-DDP suggests that the recombinogenic lesions for cisplatin are purine-purine intrastrand crosslinks.

Reference: Nowosielska A, Smith SA, Engelward BP, Marinus MG. (2006) Homologous recombination prevents methylation-induced toxicity in Escherichia coli. Nucleic Acids Res. 34, 2258-68.

Methylating agents such as N-methyl-N'-nitro-Nnitrosoguanidine (MNNG) and methyl methane sulfonate (MMS) produce a wide variety of N- and O-methylated bases in DNA, some of which can
block replication fork progression. Homologous recombination is a mechanism by which chromosome replication can proceed despite the presenceof lesions. The two major recombination pathways, RecBCD and RecFOR, which repair double-strand breaks (DSBs) and single-strand gaps respectively, are needed to protect against toxicity with the RecBCD system being more important. We find that recombination-deficient cell lines, such as recBCD recF, and ruvC recG, are as sensitive to the cytotoxic effects of MMS and MNNG as the most base excision repair (BER)-deficient (alkA tag) isogenic mutant strain. Recombination and BER-deficient double mutants (alkA tag recBCD) were more sensitive to MNNG and MMS than the single mutants suggesting that homologous recombination and BER play essential independent roles. Cells deleted for the polA (DNA polymerase I) or priA (primosome) genes are as sensitive to MMS and MNNG as alkA tag bacteria. Our results suggest that the mechanism of cytotoxicity by alkylating agents includes the necessity for homologous recombination to repair DSBs and single-strand gaps produced by DNA replication at blocking lesions or single-strand nicks resulting from AP-endonuclease action.