Single-strand breaks (SSBs) are one of the most common types of lesion arising within cells; formed by attack of genotoxic agents on the DNA, as well as enzymatically during normal cellular processes. Although the single-strand break repair (SSBR) pathway is relatively well characterised, and many components have been extensively studied in vitro, little is known of how this pathway operates in vivo when DNA is complexed with histone proteins to form chromatin. This compaction of the DNA into nucleosomal structures has the potential to inhibit repair, by sterically blocking access of repair factors to sites of DNA damage. Whilst previous studies have shown that repair of DNA double-strand breaks and UV-induced lesions are associated with alterations in chromatin structure, through covalent modification of histone proteins and nucleosome remodeling, few similar observations have been made concerning SSBR. Here, I have produced and employed mammalian cell lines stably expressing fluorescently-tagged histone proteins to analyse the dynamics of chromatin occurring upon DNA damage. Localised damage was introduced using micro-irradiation with a UV-A laser, and the histone proteins at the site of damage visualized in real-time using confocal microscopy. Through this method, I have identified a rearrangement of chromatin structure in the vicinity of DNA strand breaks in mammalian cells, resulting in a mobilization of histone proteins at the site of damage. Furthermore, I have shown that this alteration is partially dependent on the activities of both the SSBR factor poly(ADP-ribose) polymerase 1 (PARP-1), and the phosphoinositide 3-kinase-like kinase (PIKK) Ataxia telangiectasia mutated (ATM). I have examined a potential requirement for ATM in SSBR, and found no evidence of this, suggesting that the effects of PARP-1 and ATM on histone mobilization are reflective of the independent contributions of repair of single- and double-strand breaks respectively.

Nucleotide excision repair is one of the major repair pathways responsible for identifying and removing lesions in the DNA double helix. In higher eukaryotes, nucleotide excision repair is a coordinated response of over 30 proteins recruited in an ordered procession with distinct roles in the recognition, removal and repair of said lesions. A key step in the completion of the repair process is the resynthesis of the excised strand using the undamaged partner as a template. DNA polymerase kappa (polκ), a member of the Y-family, has been shown to have a role in nucleotide excision repair distinct from its traditional role in translesion synthesis. Cell lines lacking polκ showed clear defects in nucleotide excision repair and increased ultraviolet light sensitivity. Building on this established work, conserved residues were identified in the C-terminus of human polκ and mutated to alanines. Under transient expression, mutations in the ubiquitin binding domains severely impaired the recruitment to sites of damage. Cell lines defective in polκ that stably expressed these mutant polymerases showed sensitivity to ultraviolet radiation following exposure; intriguingly, this defect seems confined to the global genomic repair pathway as no substantial defect in transcription-coupled repair was observed. Following on from these observations, immunoprecipitation of the polymerase and partner proteins was investigated in an attempt to identify interactions disrupted by the mutations to the ubiquitin binding domains. These experiments indicated impairment in binding to ubiquitinated PCNA in the mutants. In further work, the recruitment of wild-type human polκ was shown to be independent of the 3' incision by the nuclease XPG during the repair process, consistent with a recently proposed model for NER.

DNA replication is a central mechanism to all forms of life. Errors occurring during DNA replication can result in mutagenesis and genome rearrangements, which can cause various diseases. In this work I have investigated the stability of direct tandem repeats (TRs) in the context of replication and replication-associated repair mechanisms. During DNA replication the replication fork encounters many obstacles, such as DNA-protein barriers, secondary DNA structures and DNA lesions. How and if replication resumes or restarts in these circumstances in order to complete genome replication is not well understood and the fidelity of replication in response to such obstacles remains unclear. I have developed TR assays to assess replication errors in the context of replication fork restart and secondary structures. The results suggest that structured DNA (G4) can cause instability of TRs in the context of normal replication and that restarted replication can be intrinsically error-prone. Surprisingly, the mutagenic effect of G4-DNA on TR stability was not elevated in the context of replication fork restart. Therefore, deletions of TRs containing G4-DNA are not more susceptible to the compromised fidelity of a restarted replication fork. Structures such as stalled replication forks can induce checkpoint responses to maintain genome stability. The stabilisation of replication forks is central in the response to replication stress. These protective mechanisms include the regulation of enzymatic activities. Mus81-Eme1 is a structure-specific endonuclease which is regulated by the DNA replication checkpoint, but has also been shown to be required for replication fork restart in certain circumstances. In collaboration with Professor Neil McDonald I analysed a novel domain identified in Mus81-Eme1. Mutagenesis of key residues deduced from the protein structure and comparison of their genetic analysis to known phenotypes of Mus81-Eme1 suggests distinct requirements for this domain.

Telomeres protect the ends of chromosomes from the activity of DNA repair machinery and provide a solution to the end-replication problem. In humans, the core protein complex located at telomeres is known as shelterin and consists of six protein subunits. Although variation is seen in the telomeric complex between species, in fission yeast the complex has notable similarities to that of humans. Separately to shelterin, the CST complex (Cdc13/Stn1/Ten1) is conserved in budding yeast, plants and mammals and is thought to negatively regulate telomerase, in addition to being required for telomere protection. However, unlike Stn1 and Ten1, Cdc13 has not yet been identified in fission yeast. Poz1 is a bridging molecule equivalent to TIN2 in human shelterin, which links the Taz1-Rap1 and the Pot1-Tpz1-Ccq1 sub-complexes, respectively bound to double- and single-stranded DNA at telomeres. Poz1 is required for the regulation of telomerase activity, and it has been hypothesised that it might do so by playing a structural role in the switching of telomeres from an open to a closed state. In this study, a reverse-2-hybrid approach was used to generate Poz1 alleles unable to interact with Rap1 or Tpz1 specifically. These alleles were subjected to phenotypic and biochemical analysis which indicated that neither individual interaction is sufficient to maintain telomere homeostasis. With telomere lengths similar to a Poz1 deletion, it is proposed that negative regulation cannot occur without the ability to form a closed complex. Given that Cdc13 is currently the only missing component in fission yeast, a second study was initiated aiming to identify a homologue by yeast-2-hybrid screening of a cDNA library, using Stn1 and Ten1 as baits. However, this approach did not yield any positive candidates. In an alternative approach, Stn1 temperature-sensitive (ts) alleles were generated and characterised. These were used to screen a genomic library for suppressors of the Stn1 ts phenotype. Several candidates were identified that require further examination while the ts allele analysis indicated that telomeres are lost in their entirety at non-permissive temperatures and that survivors of this process did so by chromosome circularisation, similar to Pot1 mutants.

Genome stability is of upmost importance to life. DNA polymerases are essential for the duplication and maintenance of the genome but they cannot themselves begin synthesis of a DNA chain, and require the activity of specialised RNA polymerases called primases. In eukaryotic cells distinct enzymes catalyse these two essential processes. This thesis contains the characterisation of coiled-coil domain containing protein (CCDC)111, a previously uncharacterised protein conserved in a broad range of unicellular and multicellular eukaryotes including humans. CCDC111 is a member of the archaeaoeukaroytic primase (AEP) superfamily and uniquely for a eukaryotic enzyme possesses both primase and polymerase activities, and was thus renamed PrimPol. The work in this thesis implicates PrimPol in the process of DNA damage tolerance, a universal mechanism by which cells complete genome duplication in spite of potentially lethal DNA damage. The first results chapters detail the essential role of a PrimPol homologue (TbPrimPol2) in the important protozoan pathogen Trypanosoma brucei. A combination of molecular, cell biology, and biochemical analyses indicate a role for TbPrimPol2 in the post-replication tolerance of endogenously occurring DNA damage using its trans-lesion DNA synthesis activity. The remaining results chapters characterise PrimPol in human cultured cells, and demonstrate that this enzyme is present in both the nucleus and mitochondria. In the nucleus PrimPol functions in the cellular tolerance of ultraviolet (UV)- induced DNA damage, and is required to protect xeroderma pigmentosum variant (XP-V) cells, deficient in the UV lesion bypass polymerase Pol !, from the cytotoxic affects of UV radiation. Together, this thesis establishes the involvement of PrimPol in DNA damage tolerance from one of the earliest diverging eukaryotic organisms to man.

Genome duplication is an essential task our cells have to achieve prior to cell division, and requires a highly specialized replication machinery to ensure it is performed in an accurate and complete manner. DNA primase and polymerases are essential components of the replisome. Primases initiate DNA replication by synthesising short RNA primers that are then elongated by faithful and processive replicative DNA polymerases. However, both exogenous and endogenous agents can damage DNA and hinder progression of the replicative machinery. Translesion synthesis DNA polymerases assist in bypassing these DNA lesions in a process called DNA damage tolerance that enables chromosomal replication to proceed in in spite of damaged templates. This thesis details the characterisation of a novel eukaryotic DNA primase, coiled-coil domain containing protein (CCDC111), a member of the Archaeo Eukaryotic Primase (AEP) superfamily. Preliminary in vitro characterisation of CCDC111 demonstrated that the recombinant protein is capable of both DNA-dependant priming and polymerase activities, which is unprecedented for a eukaryotic polymerase, and it was therefore renamed Primase-polymerase (PrimPol). The aim of this thesis was to provide one of the first cellular characterisations of PrimPol by generating a knockout of the gene in avian DT40 cells and also depleting the protein in human cells using RNAi. In vivo evidence supports the involvement of this novel polymerase in replication fork progression following replicative stress, such as exposure to UV light, but also during unperturbed DNA replication. Work in this thesis also indicates a role for PrimPol in mitochondrial DNA maintenance. Together, the data presented here establish a role for PrimPol in DNA damage tolerance in avian and human cells.

PrimPol is a recently identified member of the archaeo-eukaryotic primase (AEP) family of proteins. It possesses both primase and polymerase activities and is involved in the replication of both nuclear and mitochondrial DNA. PrimPol is predicted to possess an AEP polymerase and a UL52-like zinc finger domain. This thesis establishes the roles of these domains in the context of PrimPol's catalytic activities. Although apparently dispensable for polymerase activity, the zinc finger is essential for maintaining primase activity and also appears to play an important role in regulating the processivity and fidelity of PrimPol's extension activities. A recently study identified a PrimPol mutation (Y89D) that is potentially associated with the development of high myopia in humans. Here, the biochemical defects associated with this mutant are analysed and described. This protein variant has a significant reduction in polymerase activity. Mutational analysis suggests that the hydrophobic ring of tyrosine is important for retaining wildtype DNA extension activity. Biophysical analysis of the secondary structure and stability of this PrimPol variant suggests that this PrimPol variant has reduced α-helical content and is less stable than the wild-type protein. Finally, the interaction of PrimPol with single-stranded DNA binding protein replication protein A (RPA) is investigated. Previous studies have identified an interaction of PrimPol with RPA. Here, it is demonstrated that PrimPol has two separate RPA interaction motifs and a crystal structure is presented of one such motif in PrimPol bound to RPA that reveals the molecular basis for this interaction. Together, these studies provide molecular insights into the catalytic mechanism of PrimPol as well as some of the key intramolecular and intermolecular mechanisms of that regulate the activities of PrimPol.

Aberrant chromosome structures can promote tumors in the early stages of carcinogenesis and lead to tumor cells becoming resistant to chemotherapy, for example by changing in drug metabolism. Dicentric (containing two centromeres) and acentric (containing no centromeres) chromosomes are two abnormal chromosome structures that consider as precursors of a variety of gross chromosomal rearrangements (GCRs) generated by subsequent recombination events [1-7]. However, the mechanism of the dicentric and acentric palindromic chromosome formation and their subsequent metabolism is difficult to directly visualise. The previous results from our lab shows that replication forks stalled at a specific replication termination sequence (RTS1) can result in the formation of the dicentric and acentric palindromic chromosomes in the fission yeast Schizosaccharomyces pombe [48-52]. However, the formation of acentric and dicentric chromosomes results in a significant visability loss, due to instability and miss-segregation of the chromosomes in the yeast cells. Thus, their fate is difficult or impossible to follow. To resolve this problem, a non-essential mini-chromosome (Ch16) was developed as a novel model system in this project. The behaviour of rearranged chromosome in vivo and their subsequent fate have been visualised by integrating the lac operator (lacO) and tetracycline operator (tetO) arrays with auxotropic makers, adjacent to the RTSI locus on Ch16. The results reveal imbalanced segregation of a dicentric chromosome and subsequently undergoes a breakage event. An acentric chromosome appears to be decoupled or lost rapidly from the nucleus.

In response to DNA double stranded breaks (DSBs), mammalian cells have evolved two major repair pathways, DNA Non Homologous End Joining (NHEJ) and Homologous Recombination (HR). The majority of DSB repair in G1 and G2 phase is repaired with fast kinetics by NHEJ in a pathway that involves the core NHEJ factors: Ku, DNA-PKcs, XLF, DNA Ligase IV and XRCC4. A subset of slow repairing DSBs also requires ATM and Artemis (Riballo et al, 2004). This slow component of repair represents DSBs that reside within highly compacted regions of the genome known as heterochromatin (HC) (Goodarzi et al, 2008). ATM functions at HC to mediate relaxation by phosphorylating the HC building factor KAP-1 (Goodarzi et al, 2008). Here I provide evidence that DSBs dependent upon Artemis for their repair also reside within regions of HC. However, unlike ATM, Artemis functions downstream of the HC relaxation process. In response to DSBs, ATM phosphorylates the histone variant H2AX (γH2AX). γH2AX acts as a docking site for the localized recruitment and activation of DNA Damage Response (DDR) proteins. The expansion of γH2AX can spread over megabases of DNA. Here I have shown that highly compacted KAP-1, MeCP2 and DNMT3B enriched chromatin acts as a barrier to IR induced γH2AX expansion. In patient cells deficient for MeCP2 or DNMT3B proteins, such as Rett syndrome (MeCP2 deficient) and Immunodeficiency centromeric-instability facial-anomalies syndrome (DNMT3B deficient), ATM and Chk2 signalling is heightened, which is reflected in a hypersensitive and prolonged G2/M checkpoint arrest. These findings suggest that higher order chromatin complexity is a barrier to ATM signalling to the checkpoint machinery. In the final section of my thesis, I addressed what affect DNA damage complexity exerts on checkpoint arrest. Using exposure to heavy ion irradiation, which induces complex DSBs, I observed larger γH2AX foci and prolonged G2/M checkpoint arrest.