Telomeres cap the end of linear chromosomes and are vital for the genomic stability of eukaryotic cells. The function of telomeres depends on the interaction between the telomeric DNA and specialised telomere-bound proteins. In this study, I show that the budding yeast Rif1 (Rap1-interacting factor) exhibits unique telomere capping properties, which are distinct from its known functions. Deletion of RIF1 enhances the capping defect of the cdc13-1 mutant, whereas overexpression of RIF1 rescues the thermosensitivity of cdc13- 1 cells, and lowers the single-stranded DNA levels at damaged subtelomeres. Interestingly, Rif1 is recruited to internal damaged loci upon telomere uncapping, where it inhibits the binding of checkpoint proteins to these regions. The recruitment of Rif1 appears to be Rap1- independent as demonstrated by a mutant strain in which the Rif1-Rap1 interacting sequence was removed. In addition, Rif1 was also found essential for survival of yeast cells lacking telomeres through a checkpoint adaptation process. Furthermore, an unexpected telomere-independent role of Rif1 was discovered at an induced double strand break. Rif1 is recruited to the break site when overexpressed, where it promotes DNA repair via the nonhomologous end joining pathway. My results suggest that the budding yeast Rif1 is involved in end protection of telomeres and checkpoint adaptation. Budding yeast Rif1 may interact with the Cdc13, Stn1, and Ten1 (CST) complex and together cap the chromosome ends; scRif1 and CST may represent the functional equivalent of the vertebrate shelterin complex in the budding yeast telomeres. My study highlights the conserved function of Rif1, and implies that Rif1 may have a significant role in genomic stability and carcinogenesis in humans.

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Structural maintenance of chromosome (Smc) complexes have key functions in chromosome formation and segregation. Eukaryotes possess three essential Smc complexes: cohesin (Smc 1/3) which facilitates sister chromatid cohesion, condensin (Smc2/4), which facilitates chromosome condensation and segregation and Smc5/6, the less well-understood third complex. The Smc5/6 heterodimer interacts with the non-Smc proteins Nse1 to Nse6 to form a functional complex, implicated in DNA repair by homologous recombination (HR) and the segregation of repetitive DNA such as ribosomal DNA repeats. Hypomorphic complex mutants are HR defective and loss of Smc5/6 complex functions results in global chromosome fragmentation and missegregation.

Telomeres are specialised DNA-protein structures capping or protecting the chromosome ends fromshortening,degradation and fusions. Telomere uncapping occurs when some of the proteins associated with telomeres lose their integrity. For example in yeast a point mutation in the gene encoding the telomere binding protein Cdc13called cdc13-1,leads to conditional telomere uncapping at temperatures above 26º C. In this thesis I have utilised the cdc13-1model system to study repair after telomere uncapping. De-protection of the telomere triggers resection of the AC rich strand in 5’ to 3’ direction and formation of single stranded DNA(ssDNA). Checkpoint proteins are readily recruited to the damage and halt the cell cycle. However nossDNA re-synthesishas been observed in cdc13-1cells with uncapped telomeres. Here I will show that the ssDNA damage in cdc13-1cells recruits polymerase α,εandδand the clamp PCNA but in normal circumstancesefficientrepair is not observed. Only when telomeres are recapped the ssDNA could be re-synthesisedand this depended on the polymerase δsubunit Pol32 but did not require the non-essential subunits Dpb3 and Dpb4from polymerase ε. Interestingly, ssDNA re-synthesisat uncapped telomeres could be stimulatedthroughmild osmotic pressureand required both polymerase δand ε. Furthermore mild osmotic pressurecould also rescue cells damaged with methyl methanesulfonate but not with UV light or hydroxyurea. My data suggeststhat single stranded DNA re-synthesis may bespecifically inhibited or compete with resection when telomeres are uncapped and that osmotic pressurestimulated re-synthesisby regulating polymerases α, εand/or δ.

The mitotic spindle checkpoint is a constitutively active mechanism that protects cells from aneuploidy. Cell lines exhibit variable capacities to activate this checkpoint and rodent cells, such as CHO-K1, are well known to possess a less stringent checkpoint than human cells. Preliminary analysis of a panel of CHO-K1-human chromosome somatic cell hybrids, identified 15A (retaining human chromosome 15) as having an augmented checkpoint. Reanalysis of the two cell lines here, using both microscopy and flow cytometry, found 15A to have a higher mitotic index during an unperturbed cell cycle; but they were more unstable when their mitotic spindle checkpoint was hyper-activated with nocodazole. The complex 15A phenotype was hypothesised to result from: (1) a gene on human chromosome 15 complementing a putative CHO-K1 mitotic spindle checkpoint genetic lesion and (2) 15A cells being intrinsically "primed" for mitotic slippage by virtue of deregulation of pro-mitotic slippage mediators. The first hypothesis was addressed using a candidate gene approach. A putative CHO-K1 mitotic spindle checkpoint lesion was mapped to bubr1 at human 15q15. CHO bubr1 was cloned de novo and we have identified a putative novel heterozygous kinetochore-binding domain mutation in CHO- K1. In an attempt to validate that human BubR1 can complement the putative CHO-K1 BubR1 defect, the propensity to tolerate BubR1 overexpression was shown to be cell type/sub-clone-specific. CHO-AAS are a derivative of CHO- K1 and human BubR1 was transiently and constitutively expressed in these cells, where it increased the mitotic index and induced mitotic slippage. The second hypothesis was addressed by carrying out Western blots for markers of mitotic spindle checkpoint activation, mitotic slippage and apoptosis in basal and nocodazole treated CHO-K1 and 15A cells. Most notably, TAp73 protein (a transcriptionally active p53-family member protein) is specifically and constitutively highly upregulated in 15A under basal conditions. Downstream TAp73 targets, such as Cdk inhibitors p21 and p57, create a pro-mitotic slippage milieu in 15A. TAp73 was shown to be post-transcriptionally upregulated in 15A; to be localised in the nucleus and to be stabilised independent of a canonical DNA- damage response (as evidenced by Western blotting for y-H2AX and PML proteins). Ectopic TAp73 overexpression in CHO-K1 induced apoptosis. Thus, the highly pro-apoptotic TAp73 is held in check in 15A. This warrants further investigation that may elucidate novel mechanisms for p73 regulation.

The overall aim of this project is to study the control of hCD2 gene expression and how different parts of the gene contribute to its regulation. This was achieved by searching for novel regulatory elements within the hCD2 gene whose function could be addressed by deleting them using an in vivo Cre/loxP deletion strategy. For this purpose it was necessary to develop tools that would, in future, enable the in vivo tissue-specific deletion of these identified sequences utilising this approach. To identify novel regulatory elements within the hCD2 gene a number of different methods were used. Previously intron 4 has been shown to confer elevated levels of expression on hCD2 transgenes, however its function has never been studied in the context of variegation. Therefore, a construct including intron 4 and the truncated LCR was generated. This vector allowed elevated levels of hCD2 expression without conferring protection against PEV in transgenic mice, thus confirming that intron 4 could not compensate for a debilitated LCR. In addition, it was shown that decreasing transgene copy number whilst retaining the same site of integration affected hCD2 variegation in a manner that was dependant upon the site of integration. Therefore, we established that the inclusion of intron 4 in transgenes would be used for variegation studies whilst ensuring high levels of expression.

In most societies, surnames are passed down from fathers to sons, just like the Y chromosome. It follows that, theoretically, men sharing the same surnames would also be expected to share related Y chromosomes. Previous investigations have explored such relationships but most data has been collected so far only from the British Isles. In order to provide additional in sights into the con-elation between surnames and Y chromosomes, this study focuses on the Spanish population and investigates Y chromosome SNP/STR variation by analysing a total of 1,766 DNA samples from unrelated Spanish male volunteers belonging to 37 surnames and 355 controls. The results suggest that the degree of coancestry within surnames is highly dependent on surname frequency. Within-surname genetic variation, as measured by different statistics, con-elates well with surname frequency, though a few exceptions are found. In addition, geographic distance between the individuals' place of origin influences Significantly the con-elation between Y chromosome and surnames: men with the same surname tend to have more similar Y chromosomes if their paternal grandfathers were born geographically close to each other. Therefore, it seems that Y chromosome coancestry within surnames is as much about surname frequency as it is about geographical proximity.