DNA Damage Recognition Proteins and Their Involvement in Cisplatin Resistance

cis-Diamminedichloroplatinum(II) (CDDP) is a chemotherapeutic agent widely used in the treatment of various types of cancer. Its mechanism of cytotoxicity is unclear although it is believed that DNA is the critical target. CDDP binds to DNA forming a variety of adducts including intrastrand adducts, interstrand adducts, monofunctional adducts and DNA-protein crosslinks. This thesis presents evidence that there are protein(s) present in mammalian cells which recognise CDDP-damaged DNA, To assay for these DNA damage recognition proteins (DDRPs) conditions for two very separate assays were developed. The gel mobility shift assay, which detects protein complexes under non-denaturing conditions, identified two retardation complexes which bound to CDDP damaged DNA in human, murine and feline tumour cell extracts. Binding of these complexes is shown to CDDP treated oligonucleotide of 54 base pairs but not to a CDDP treated oligonucleotide of 27 base pairs, therefore suggesting binding is dependent on having normal DNA duplex. The other system used in the detection of the DDRPs is the South-Western assay. This allowed the detection of proteins of sizes 25, 50, 100KD binding to CDDP treated DNA. The proteins in the South-Western system are run under denaturing conditions. It is not entirely clear as to whether the proteins detected in both systems are the same or whether they represent entirely different species. CDDP has been reported to bind to DNA and cause areas of singlestrandedness around the adducts. The results presented in this thesis demonstrate that the 50KD and 100KD DDRP which bind to CDDP treated double-stranded DNA may also have an affinity for single-stranded DNA. The 25KD DDRP, however, only recognises double-stranded DNA treated with CDDP suggesting that it is recognising the CDDP adducts and not the areas of single-strandedness generated around the adducts. Resistance to CDDP proves a major problem area in treatment regimes. Many cell lines resistant to CDDP have been derived in vitro by multiple exposures to the drug. Many mechanisms of resistance to CDDP have been suggested from these lines. If a role of the DDRPs was to process damage in the DNA then cell lines resistant to CDDP may show an increase in expression of the DDRPs. This thesis presents evidence that an ovarian tumour cell line resistant to CDDP in comparison to its parental line shows an increase in the binding to the 50KD and 100KD DDRPs. Work in chapter 5 presents the isolation of CDDP resistant cell lines, by acute exposure to the drug, with an increase of up to seven fold resistance levels. Evidence is presented for the resistant clones being of a mutational origin. Resistant variants occur at a frequency of 3.2x10e-6 per viable cell. This frequency can be increased to 3.4x10e-5 by treatment of the cells with the chemical mutagen ethyl methane sulphonate, EMS. The CDDP resistant phenotype is maintained after six months growth in drug free medium. This single step selection may provide clones which are more clinically relevant than the lines isolated by multiple exposures to CDDP. They may therefore provide a superior model for the study of drug resistance mechanisms to CDDP. However examination of the DDRPs showed no detectable difference in the resistant clones derived from the A2780 human ovarian tumour cell line. The thesis therefore presents evidence of the existence of DDRPs in mammalian cells. The role of these damage recognition proteins will be discussed

The roles of transcription factors in Nucleotide excision repair in yeast

Nucleotide excision repair (NER) is a conserved DNA repair mechanism capable of removing a variety of helix-distorting lesions, such as UV-induced cyclobutane pyrimidine dimers (CPDs). NER can be grouped into two pathways: global genomic NER (GGR), which refers to repair throughout the genome, and transcription coupled NER (TCR), which refers to a repair mechanism that is dedicated to the transcribed strand (TS) of actively transcribed genes. In yeast S. cerevisiae, Rad7, Rad16, and Elc1 are specifically required for GGR. TCR is believed to be initiated by RNA polymerase II (Pol II) stalled at a lesion in the TS of a gene. Rad26, the yeast homolog of the human CSB protein, and RPB9, a nonessential subunit of Pol II, play important roles in TCR. However, the exact mechanisms of NER in eukaryotic cells are still elusive. By using yeast S. cerevisiae as a model organism, this dissertation focused on the functional mechanisms of transcription factor Tfb5, transcription elongation factors Spt4 and Spt5, and the putative yeast transcription repair coupling factor (TRCF) Rad26 in NER, especially in TCR pathway. Tfb5, the tenth subunit of the transcription/repair factor TFIIH, is implicated in one group of the human syndrome trichothiodystrophy (TTD). We found that Tfb5 plays different roles in different NER pathways in yeast. Tfb5 is essential for GGR and Rpb9 mediated TCR. However, Tfb5 is partially dispensable for Rad26 mediated TCR, especially in GGR deficient cells. Spt4 and its interacting partner Spt5 cooperatively suppress TCR only in the absence of Rad26, regardless of the presence of Rpb9. The phosphorylation of C-terminal repeat (CTR) domain of Spt5 by the Bur kinase plays an important role in the suppression. Immunoprecipitation results indicate that Rad26 dynamically associates with Pol II and restrains the binding of Spt4/Spt5 to Pol II. ATPase activity of Rad26 is required for facilitating TCR and for restraining the binding of Spt4/Spt5 to Pol II. Finally, we proposed that Rad26 enhances TCR by restraining the binding of suppressors Spt4/Spt5 to Pol II. These findings provide new insights into the functional mechanisms of Tfb5, Spt4/Spt5 and Rad26 in NER, especially in TCR

Investigation of Novel Functions for DNA Damage Response and Repair Proteins in Escherichia coli and Humans

Endogenous and exogenous agents that can damage DNA are a constant threat to genome stability in all living cells. In response, cells have evolved an array of mechanisms to repair DNA damage or to eliminate the cells damaged beyond repair. One of these mechanisms is nucleotide excision repair (NER) which is the major repair pathway responsible for removing a wide variety of bulky DNA lesions. Deficiency, or mutation, in one or several of the NER repair proteins is responsible for many diseases, including cancer. Prokaryotic NER involves only three proteins to recognize and incise a damaged site, while eukaryotic NER requires more than 25 proteins to efficiently recognize and incise a damaged site. XPC-RAD23B (XPC) is the damage recognition factor in eukaryotic global genome NER. The association rate of XPC to damaged DNA has been extensively studied; however, our data suggests that the dissociation of the XPC-DNA complex is the rate-limiting step in NER. The factor that verifies DNA-damage downstream of XPC is XPA. XPA also has been implicated in binding of ds-ssDNA junctions and has been found to bind at or near double-strand break sites in the premature aging syndrome Hutchinson-Gilford progeria (HGPS). This role for XPA is outside of its known function in NER and suggests that XPA may bind at collapsed replication forks in HGPS that are unprotected due to a lack of binding by replication proteins. Along with XPC and XPA, ataxia telangiectasia and Rad3-related (ATR) is activated in response to DNA damage and initiates the cell cycle checkpoint pathway to rescue cells from genomic instability. We found that ATR functions outside of its known role in the checkpoint signaling cascade. Our data demonstrate that ATR can rescue cells from apoptosis by inhibiting cytochrome c release at the mitochondria though direct interaction with the outer mitochondrial membrane and the proapoptotic protein tBid. The role of ATR in apoptosis is regulated by Pin1, which can change the structure of ATR at the backbone level. All of the results presented here suggest novel roles for DNA repair proteins in the maintenance of genome stability

Processing of UV-induced DNA damage in human skin cells

It was the aim of the experimental work presented in this thesis to identify -in mammalian cells- the lesions responsible for UV-induced biological effects, such as cell-killing, to study the relation between the removal of lesions and the occurrence of repair synthesis of DNA ("unscheduled DNA synthesis") and to investigate the possible role and practical significance of photoreactivation in human