No description available.

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No description available.

Genomic integrity is preserved by checkpoints, which are signal transduction pathways that serve to delay cell cycle progression in the presence of DNA damage or replication stress. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1) complex is a proliferating cell nuclear antigen (PCNA)-like clamp that is loaded onto DNA at structures resulting from damage, and is important for initiating and maintaining checkpoint signaling. Rad9 possesses a C-terminal tail unrelated to PCNA that is phosphorylated constitutively and in response to cell cycle position and DNA damage. Previous studies have identified tousled-like kinase 1 (TLK1) as a kinase that may modify Rad9. This thesis establishes that Rad9 is indeed phosphorylated in a TLK-dependent manner in vitro and in vivo, and that T355 within the C-terminal tail is the primary targeted residue. Phosphorylation of Rad9 at T355 is quickly reduced upon exposure to ionizing radiation before returning to baseline later in the damage response. In addition, TLK1 and Rad9 were shown to interact constitutively, and this interaction is enhanced in chromatin-bound Rad9 at later stages of the damage response. Furthermore, this thesis demonstrates that TLK1 is required for progression through S-phase in normally cycling cells, and that depletion of TLK1 results in a prolonged G2/M arrest upon exposure to ionizing radiation, a phenotype that is mimicked by over-expression of a Rad9-T355A mutant. Given that TLK1 is transiently inactivated upon phosphorylation by Chk1 in response to DNA damage, this work proposes that TLK1 and Chk1 act in concert to modulate the phosphorylation status of Rad9, which in turn plays a role in regulating the DNA damage response. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2013-12-24 10:31:57.987

DNA is a highly unstable molecule. Endogenous souces of DNA damage, such as reactive oxygen species (ROS), can cause DNA damage and it has been estimated that 20000 lesions occur in a cell per day. BER is the major pathway for the repair of these lesions and therefore maintains genome stability, thus preventing the development of human diseases such as neurodegenerative diseases and cancer. Therefore, if BER cannot accomplish the repair, accumulation of DNA damage occurs, triggering different cellular responses, such as cell cycle delay and senescence. The ARF tumour suppressor protein, the gene of which is frequently mutated in many human cancers, plays an important role in the cellular stress response by orchestrating upregulation of p53 protein. Moreover, ARF expression is upregulated in senescent cells, suggesting that ARF induction might be triggerred in response to persistent DNA damage. Although ARF has been reported to be important in the regulation of proteins involved in the DNA damage response, its role is still controversial. Here, it has been shown that ARF gene transcription is induced by DNA strand breaks (SBs) and that ARF protein accumulates in response to persistent DNA damage generated by disabling BER. These data suggest that PARP1-dependent poly(ADP-ribose) synthesis at the sites of SBs initiates DNA damage signal transduction by reducing the cellular concentration of NAD⁺, thus inhibiting SIRT1 activity and consequently activating E2F1-dependent ARF transcription. These findings suggest a vital role for ARF in DNA damage signalling, and furthermore explain the critical requirement for ARF inactivation in cancer cells, which are frequently deficient in DNA repair and accumulate DNA damage.

DNA repair mechanisms constitute an essential cellular response to DNA damage arising either from metabolic processes or from environmental sources such as ultraviolet radiation. Repair of these lesions may be via direct reversal, or by processes such as nucleotide excision repair (NER), a coordinated pathway in which lesions and the surrounding nucleotides are excised and replaced via DNA resynthesis. The importance of repair is illustrated by human disease states such as xeroderma pigmentosum and Cockayne's syndrome which result from defects in the NER system arising from mutations in XP- genes or XP- and CS- genes respectively Little detail is known of DNA damage repair processes in plants, despite the economic and ecological importance of these organisms. This study aimed to expand our knowledge of the process of NER in plants, largely via a polymerase chain reaction (PCR)-based approach involving amplification, cloning and characterisation of plant genomic DNA and cDNA. Homologues of the NER components XPF/RAD1 and XPD/RAD3 were isolated as both genomic and complete cDNA sequences from the model dicotyledonous plant Arabidopsis thaliana. The sequence of the 3'-untranslated region of atXPD was also determined. Comparison of genomic and cDNA sequences allowed a detailed analysis of gene structures, including details of intron/exon processing. Variable transcript processing to produce three distinct transcripts was found in the case of atXPF. In an attempt to validate the proposed homologous function of these cDNAs, assays to test complementation of resistance to ultraviolet radiation in the relevant yeast mutants were performed. Despite extensive amino acid sequence conservation, neither plant cDNA was able to restore UV-resistance. As the yeast RAD3 gene product is also involved in vivo in transcription, and so is required for viability, the atXPD cDNA was tested in a complementation assay for this function in an appropriate yeast mutant. The plant cDNA was found to substantially increase the viability of the yeast mutant. The structural and functional significance of these results is discussed comparatively with reference to yeast, human and other known homologues. Other putative NER homologues were identified in A. thaliana database sequences, including those of ERCC1/RAD10 and XPG/ERCC5/RAD2, and are now the subjects of ongoing investigations. This study also describes preliminary investigations of putative REVS and RAD30 translesion synthesis genes from A. thaliana.

Graduation date: 2003

Here the ciliate protozoa Tetrahymena thermophila was used as a model system to study the DNA damage response. Tetrahymena enclose nuclear dimorphism, a polyploid somatic macronucleus (MAC), which is transcriptionally active and maintains vegetative growth, and a diploid germline micronucleus (MIC) responsible for the transmition of genetic information during conjugation. Previous studies have identified Tif1p, a novel protein involved in the regulation of rDNA replication in Tetrahymena. TIF1 hypomorphic strains acquire spontaneous DNA damage during vegetative cell cycle and are hypersensitive to DNA damaging agents. TIF1-deficient strains acquire DNA damage in both nuclear compartments, suggesting a global role of Tif1p in the maintenance of genomic stability. In my dissertation research, I studied the role of Tif1p during the cell cycle progression. To this end, I generated tagged-Tif1p strains, which revealed that the subcellular localization of Tif1p is dynamic throughout the cell cycle. However, the addition of epitope tag to this protein generated phenotypes analogous to ones observed in a TIF1-deficient strain. This suggested that the addition of epitope tag to Tif1p severely affects the properties of Tif1p and hence the overall integrity of the cell. To overcome these limitations, a peptide antibody specific to Tif1p was generated to study the endogenous protein. This work revealed that the abundance of Tif1p protein is not cell cycle regulated and that Tif1p is absent in starved cells. Furthermore, the specific binding of TIf1p to rDNA minichromosome was studied during vegetative cell cycles. Chromatin immunoprecipitation studies revealed that the specific binding of Tif1p extends beyond the cis-acting determinant of replication present at the rDNA origin and promoter. This suggests that coding regions may be targeted for the binding of Tif1p to previously uncharacterized sequences, and that Tif1p preferentially localizes on the rDNA minichromosome. I also studied the induction of DNA damage response, demonstrating that Tetrahymena activates a checkpoint response mediated by an ATR-like pathway. Studies with a hypomorphic TIF1 strain revealed that Tif1p mediates proper activation of the DNA damage response. Further characterization of the response to genotoxic agents showed that Tetrahymena is able to activate a G1/S and intra-S phase DNA damage response. The results presented here suggest that a caffeine-dependent checkpoint activator protein modulates the response to DNA damage. In addition, a subunit of the replicative helicase, Mcm6p, is directly affected by the induction of DNA damage. This suggests that Tetrahymena uses a novel mechanism to halt the progression of DNA replication forks during genotoxic stress through degradation of Mcm6p.