The Role of DNA Mismatch Repair in Cellular Responses to DNA Damage and Drug Resistance

Durant, Stephen Thomas (1999) The Role of DNA Mismatch Repair in Cellular Responses to DNA Damage and Drug Resistance. PhD thesis, University of Glasgow.

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DNA mismatch repair (MMR) is a system of evolutionary conserved DNA surveillance proteins that maintain the integrity of the genome by correcting errors of DNA replication and by regulating DNA excision and recombinational repair processes. Loss of human MMR is associated with microsatellite instability (MI), a mutator phenotype, and hereditary as well as sporadic cancers. Cells deficient in MMR display resistance to the cytotoxic effects of an expanding list of DNA-damaging agents such as DNA methylating, platinating and antimetabolite drugs, many of which are routinely used in the treatment of cancer. MMR deficiency has also been linked to defects in G2 cell cycle arrest in response to DNA damage. To explore the role that MMR has in cellular responses to DNA damage in isogenic strains of S. cerevisiae, individual disruptions in the MMR genes MSH2, MSH3, MSH6, MLH1, K4LH2 and PMS1 were tested for cytotoxic and cell cycle responses to various DNA damaging agents. A significant 2.6 - 5.6-fold increase (P<0.05) in clonogenic resistance to a 24 hour exposure of 1mM cisplatin was seen in all mutants except for pmsl, compared to wild type. All mutants, but not mlh2, displayed a 10-30-fold increased forward mutation frequency, indicative of loss of MMR function. These results indicate that specific components of MMR, but not necessarily MMR per se, are involved in conferring sensitivity to cisplatin. Re-introducing ScMLHl back into the mlh1 mutant using a high copy yeast expression vector restored cisplatin sensitivity to levels greater than wild type. Strains null for the recombinational repair gene RAD52 and the recombination/nucleotide excision repair gene RAD1 were hypersensitive to cisplatin. Mlh1, msh2 and mlh2 strains in rad52 and rad1 backgrounds were as sensitive as single rad52 and rad1 strains indicating that the presence of these recombination/excision repair proteins is required for the MMR-deficient-induced resistance phenotype. Wild type cells grown to confluence and hence under growth inhibitory conditions were more resistant to killing by cisplatin than logarithmically growing cells, whereas no difference was seen in the msh2 mutants. This suggests a requirement for DNA replication for the MMR-dependent cytotoxic response. A model in which MMR acts to inhibit recombinational bypass of cisplatin-induced DNA adducts is described. Cell cycle analysis of these strains indicated that mlh1 and msh2 disruption lead to an early release from G2-M arrest in response to a 1 hour, 4mM exposure to cisplatin. MMR mutants exposed to 100gy ionising irradiation, after a transiently quicker entry into G2-M, did not display any significant difference in arrest pattern or cytotoxicity compared with wild type. These results suggest that MMR in budding yeast functions in a cisplatin-specific DNA damage response pathway that leads to a prolonged G2 cell cycle arrest response. A model is described in which MMR induces S. cerevisiae cell cycle checkpoint arrest in response to persistent cisplatin-induced DNA lesions and/or replication inhibition.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Bob Brown
Keywords: Biochemistry
Date of Award: 1999
Depositing User: Enlighten Team
Unique ID: glathesis:1999-75522
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 19:34
Last Modified: 19 Nov 2019 19:34

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