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Efficient acquisition of the genes (hsdR, M and S) that specify EcoKI and EcoAI, representatives of two families of type I restriction and modification (R-M) systems, was shown to require a product of an unknown gene hsdC. The hsdC mutant is shown to have a mutation in clpX. ClpP, the components of ClpXP protease, are necessary for the efficient transmission of the hsd genes by conjugation, transformation and P1 transduction. Inactivation of clpX leads to a bigger barrier than a similar mutation in clpP consistent with a chaperone activity of ClpX in the absence of ClpP. The establishment of the modification activity of EcoKI is not dependent on clpX and takes about 12 generations to reach its maximal activity in methylating incoming phage DNA. This lag probably reflects the time necessary to complete the methylation of bacterial chromosomes. Modification, once established, has been assumed to provide adequate protection against a resident restriction system. However, unmodified targets may be generated in the DNA of an hsd⁺ bacterium as the result of replication errors or recombinant-dependent repair. The presence of unmodified target sequences for type I restriction-modification systems on bacterial chromosomes does not influence the survival of hsd⁺ bacteria due to ClpXP- dependent regulation of the endonuclease activity. HsdR, the polypeptide of the R-M complex essential for restriction but not modification, is degraded in the presence of ClpXP and therefore the bacteria show a temporary drop in restriction activity, referred to as restriction alleviation. The delayed detection of restriction activity followed by the establishment of a new specificity can be considered as a case of restriction alleviation. The data obtained support a model in which the HsdR component of a type I restriction endonuclease becomes a substrate for proteolysis after the endonuclease has bound to unmodified target sequences on the chromosome, but before completion of the pathway that would result in DNA breakage. It remains unclear how the restriction-modification systems distinguish between unmethylated host and foreign DNA. The latter is degraded while the former is protected from cleavage by ClpXP-dependent proteolysis of HsdR.

Telomeres are the ends of linear eukaryotic chromosomes essential for their stable maintenance. The conventional replication machinery is not able to copy linear DNA molecules to the very end. To overcome "the end replication problem" most eukaryotes rely on telomerase to maintain telomeres to extend back the shortened telomeres and maintain the protection of chromosome ends from fusions and degradation. However, in most stem and somatic cells in humans, the levels of telomerase are developmentally downregulated. The residual telomerase activity is insufficient to compensate for telomere attrition during replication. As a result, telomeres can become critically short, which would trigger activation of the DNA damage response and cell cycle arrest, followed by either senescence or apoptosis. This mechanism is considered a cell proliferation barrier evolved to suppress tumour growth. In S.cerevisiae, telomerase is constitutively expressed, but the telomere shortening due to the loss of telomerase components also leads to a decrease in the proliferation capacity and frequent arrests. Here, I have engineered yeast cells with telomerase insufficiency (TI) to create a yeast model of human cell populations with downregulated telomerase. Using fluorescent microscopy, I followed the dynamics of the nucleus during mitosis and detected frequent nuclear missegregations in cells with TI. The genetic analysis of the missegregations demonstrated that the observed aberrant mitotic events were not caused by NHEJ or RAD51-dependent recombination at critically short telomeres, but they were dependent on the presence of the DNA damage checkpoint sensor Mec1, and its mediator Rad9. Similar nuclear missegregations were also observed in cells with the HO endonuclease-induced double-stranded breaks (DSBs) and in response to the DSB-inducing drug phleomycin. Therefore, activation of the DNA damage response correlated with the increased frequency of aberrant mitoses. In response to DNA damage, two largely independent parallel pathways of the DNA damage checkpoint mediated by Chk1 and Rad53 are activated to prevent anaphase entry and mitotic exit. This study reports that mutation or deletion of components of these pathways and subsequently manipulating the efficiency of cohesion cleavage or inhibition of the mitotic exit network affected the probability of a cell cycle arrest to result in aberrant mitosis. To summarise, these results suggest that nuclear divisions after the activation of the DNA damage checkpoint have an increased frequency of nuclear missegregations. These missegregations might be caused by an altered order of the key events during mitosis, where a delay in cohesin cleavage prevents the timely separation of sister chromatids and leads to the majority of DNA being inherited by either a mother or a daughter cell.

Eukaryotes maintain their genomes in the form of linear chromosomes. Because of the inability of the replication machinery to copy linear DNA molecules to the very end, the chromosomes were predicted to become shorter with every round of replication. This phenomenon was called "the end replication problem" by Watson and Olovnikov. Later, it has been discovered that eukaryotes have evolved telomeres, non-coding DNA repeats at the chromosomal ends, and telomerase, a ribonucleoprotein that extends the telomeres to overcome "the end replication problem". Telomerase is known to be downregulated in humans through the development resulting in progressive telomere shortening and replicative senescence with age. This is believed to be one of the major tumour suppressor mechanisms. However, most cancer cells reactivate telomerase to become immortal. Thus, understating telomerase regulation is one of the central questions in the field of cancer biology. Telomerase complex formation involves several component maturation and assembly steps. In Saccharomyces cerevisiae, the steady-state levels of the catalytic subunit Est2 are significantly reduced when its interaction with the telomerase RNA component TLC1 is impaired. I have found that Est2 not bound to TLC1 undergoes degradation in a proteasome-dependent manner. Loss of Tom1, an E3-ubiquitin ligase, leads to an increase in the Est2 levels accompanied by a decrease in the Est2 degradation rate. Consistent with these findings, tom1 mutants have longer telomeres. Furthermore, Tom1 physically interacts with Est2, specifically through recognizing its RNA binding domain. Disruption of TOM1 does inhibit proteolysis of Est2 but does not stop it completely, suggesting the existence of an additional, Tom1-independent degradation pathway of Est2. Interestingly, the Est2 levels are reduced in cells grown at elevated temperatures. This decrease is caused by the temperature-triggered degradation of Est2. This degradation resembles proteolysis through the protein quality control system, however, the exact regulator of this mechanism is yet unknown. I propose that regulation of the telomerase through the proteolysis contributes to telomere length homeostasis indirectly through controlling the levels of Est2. The human Est2 homologue hTERT is also degraded in a proteasome-dependent manner. Thus, the proteolysis-dependent regulation of the telomerase might be evolutionarily conserved. RNA-free hTERT is believed to have extra-telomeric roles and this regulation might balance its telomeric and extra-telomeric functions.

Genome stability in eukaryotic cells relies heavily on their ability to differentiate between telomeres - natural ends of linear chromosomes - and double strand breaks (DSBs), pathological lesions which can occur throughout the stretch of chromosomal DNA. The former are to be protected from fusions, recombination and recognition by DNA damage response, whereas the latter are to be detected by the checkpoint system and repaired by end joining or homologous recombination. Due to the end replication problem, continually dividing cells are confronted with an additional necessity to maintain stable telomere length, which in most eukaryotes is fulfilled by telomerase. Telomerase is downregulated in human somatic cells to limit their replicative capacity and prevent malignization, forcing pre-malignant cells to seek ways of re-establishing telomere length homeostasis. Most cancers perform it by reactivating telomerase. Recently, Makovets group found a yeast model for telomerase reactivation through aneuploidy in cells with temperature-induced telomerase insufficiency. Given that aneuploidy is a common feature of cancers, a deeper mechanistic understanding of aneuploidy-driven telomerase reactivation in yeast may shed more light on cancer telomere biology. In telomerase-negative cancers telomere attrition is counteracted by alternative lengthening of telomeres (ALT). ALT is related to one of DSB repair pathways - break-induced replication (BIR), which operates on single-ended DSBs, such as those originating from broken replication forks. Phosphorylated Pif1 helicase has been reported to be essential for BIR, but the molecular mechanism of this requirement has not been ascertained. We found that the need for phosphorylated Pif1 in BIR is alleviated in yeast expressing an Nterminally truncated Dna2 nuclease/helicase. In my work I aim to gain insights into how yeast solve problems related to both natural chromosomal ends and DSBs by investigating mechanisms underlying aneuploidy-dependent telomerase reactivation in yeast cells with temperature-induced telomerase insufficiency and by studying the genetic interaction between Dna2 and Pif1 in BIR.

Senescence is defined as a permanent and irreversible cell cycle arrest. The initiating stimuli can be diverse, such as oncogene expression, reactive oxygen species and critically short telomeres. Irrespective of the nature of the trigger, p53-dependent DNA damage response (DDR) is initiated, leading to cell cycle arrest in G1. A prolonged DDR activation can further reinforce the arrest through the activation of the cyclin-dependent kinase inhibitor p16. The inability of the semi-conservative DNA replication machinery to completely copy the ends of linear eukaryotic chromosomes results in a gradual telomere shortening that is counteracted by telomerase, a ribonucleoprotein which can add telomeric repeats to the 3’ end of a chromosome. However, during human embryonic development, telomerase is downregulated through decreased expression of the telomerase catalytic subunit hTERT. Consequently, human somatic cells undergo progressive telomere shortening that will ultimately lead to eroded telomeres being recognised by the DDR and induction of replicative senescence. Reconstitution of hTERT in several human somatic cells has been shown to prevent replicative senescence. During cancer development, cell immortalisation requires escaping senescence and telomerase reactivation is the prevalent route to this end. However, while it is clear that the presence of telomerase can counteract entry into the senescence, it is not known if, once senescence has been established, telomerase could drive cells out of the arrest. In this work, I address the reversibility of senescence triggered by critically short telomeres by: 1) reactivating telomerase expression in senescent cells; 2) reactivating expression and tethering telomerase to telomeres during senescence; and 3) reactivating telomerase expression together with transient DDR inhibition. By taking advantage of telomerase fused to a conditional degron, I have tested if telomerase stabilisation in senescent MRC5 human fibroblasts could bypass the arrest. My results show that the presence of telomerase was not sufficient to overcome senescence. Since it has been shown that yeast telomerase can only gain access to telomeres during DNA replication, one possibility is that telomerase reactivated in senescent cells was unable to localise to telomeres. To address this possibility, I have expressed an hPOT1-hTERT fusion to tether telomerase to telomeres irrespective of the cell cycle stage. In a parallel approach, I have set up a system to allow senescent cells to enter the S-phase by transient inhibition of DDR, by IPTG-inducible knock-down of p53 and p16. This study contributes to improving our understanding of molecular mechanisms of senescence and its reversibility as a possible approach for the therapy of agerelated diseases.

Proteins are the ultimate effectors of biological mechanisms and are involved in every aspect of cellular life. The functional properties of proteins depend on their three-dimensional structure. Indeed, proteins are not rigid entities and internal motions, on a wide range of timescales and distances, are necessary to accomplish a specific function. While certain proteins adopt a compact conformation and undergo small-scale rearrangements, others are more flexible and withstand more dramatic movements. Proteins that exhibit a regulatory role function by binding multiple partners, such as small molecules, DNA, RNA, other proteins. Increased levels of flexibility favour this binding promiscuity. A long-standing goal in molecular biology has been the development of new methods to enable the determination of three-dimensional structures of proteins that exhibit a high level of dynamic complexity. In fact, it is becoming clearer every day that individual structural techniques have several limitations. An integrative structural biology approach provides the tools to decipher the dynamic configuration of proteins by combining information from multiple sources, including biochemical, bioinformatics and biophysical methods. In this thesis, an integrated approach is used to structurally characterize two extremely different nucleic acids binding proteins, the human Staufen1 protein and the archaeal Pyrococcus abyssi MCM complex. In this study, I show that an integrated structural biology approach is not only essential to determine the architecture of small, flexible proteins, which are traditionally difficult targets for conventional approaches, but it is also beneficial to understand the dynamic behavior of large, more compact macromolecular complexes.

Stable maintenance and transmission of the genetic material requires tight coordination between DNA replication and DNA repair. One of the factors that couples DNA replication and DNA repair is proliferative cell nuclear antigen (PCNA). PCNA is a homotrimeric complex that encircles DNA and plays a structural role interacting with different enzymes, tethering them to DNA and promoting their catalytic activity. PCNA is heavily post-translationally regulated, which was shown to change its affinity to the interacting partners and therefore to confer differential recruitment of the required proteins to the DNA substrates. Pif1 family of DNA helicases in Saccharomyces cerevisiae is composed of the two paralogues Pif1 and Rrm3. In addition to the overlapping functions during DNA replication, Pif1 and Rrm3 have certain distinct roles. Rrm3 is required for DNA replication through the “hard-to-replicate” regions, such as rDNA repeats, telomeres and origins of replication. The unique functions of Pif1 involve telomerase inhibition at telomeres and at DNA double-strand breaks (DSBs) and promotion of break induced replication (BIR). The underlying mechanism that defines functional specialisation of Pif1 family helicases is not understood. Both Pif1 and Rrm3 were shown earlier to physically interact with PCNA. The experiment presented here shows that, in addition to the previously reported C-terminal PCNA interacting peptides (PIP1 and PIP2), the N-terminus of Pif1 contains putative PCNA interacting motifs (PIP3 and SIM). The Pif1 roles in DNA replication, BIR and telomerase inhibition require different PCNA-interacting motifs. In addition, Pif1 recruitment to stalled forks and DSBs was PCNAdependent. Despite the fact that the Pif1-PCNA complex was shown to be required for Pol δ-mediated DNA synthesis during BIR, it is still not clear if other stages of this DNA repair pathway are Pif1-dependent. This study shows that the broken strand invasion and the initiation of DNA synthesis during BIR occur independently of Pif1, however both the long-range DNA synthesis and re-synthesis of the resected strand require Pif1 and its previously reported Rad53/Dun1-dependent phosphorylation. Based on the published data, the interaction between Rrm3 and PCNA occurs via the N-terminally located PCNA interacting motif (PIP1 in Rrm3). The experiment presented here shows that PIP1 in Rrm3 was not required for DNA replication through the sites that require Rrm3 helicase. In contrast, replication associated function of Rrm3 required the C-terminally located putative PCNA interacting motif (PIP2 in Rrm3), homologous to the previously reported C-terminal PCNA interacting motif in Pif1 (PIP2 in Pif1). Overall, this work provides a systematic characterisation of the newly identified and previously reported pif1 and rrm3 alleles and shows that different functions of Pif1 can be distinguished by the genetic requirements for its PCNAinteracting elements. Based on the obtained data, I would like to hypothesise that differential recruitment of Pif1 helicases via interaction with PCNA could be the reason of the functional specialisation of Pif1 and Rrm3.

Faithful DNA replication is essential for the maintenance of genetic information. This complex process consists of 3 steps: initiation, elongation and termination. Although the first two steps are quite well understood in both eukaryotes and prokaryotes, many aspects of the termination of replication remain unclear. Escherichia coli is an ideal organism to study termination of DNA replication. In E. coli, DNA replication starts by bidirectional firing of two replication forks from a unique origin and terminates when those forks collide in the terminus region of the circular chromosome. The terminus region is flanked by specific ter sequences, which ensure that termination of replication occurs within specific boundaries. Due to the circularity of the E. coli chromosome, once the replication is finished the dimers can be formed. To resolve the dimers, the dif sequences are aligned together and two chromosomes are then separated into two daughter cells. Previous members of Prof. Leach laboratory have observed a stimulation of both double-strand break repair (DSBR) and DNA over-replication in the terminus region when DSBR was induced in the lacZ locus, half way between the origin and the terminus. In this work, I propose that these two phenomena, elevated levels of DSBR and DNA over-replication, are linked to each other. I confirm that the DSBs arise from the dif site and that the dif site is the source of DNA over-replication in the terminus. My results suggest that an attempted DSBR at dif leads to over-replication between terA and terB. Here, using next generation sequencing methods, I show that TopoIV and TopoIII topoisomerases introduce breaks in chromosome dimers that were not resolved by the XerCD/dif system, leading to DSBR and DNA over-replication.

DNA double strand breaks (DSBs), which may occur during DNA replication or due to the action of genotoxic agents, are extremely dangerous DNA lesions as they can cause chromosomal rearrangements and cell death. Therefore, accurate DSB repair is vital for genome stability and cell survival. Two main mechanisms serve to repair DNA DSBs: non-homologous end joining, which re-ligates DNA ends together, and homologous recombination (HR), which restores broken DNA using homologous sequence as a template for repair. One-ended DSBs are a subject for the specialised HR-dependent repair pathway known as break-induced replication (BIR). At low frequency, DNA breaks can also be healed by telomerase, which normally extends telomeres at natural chromosome ends, but may also add de novo telomeres to DSBs due to their similarity to chromosome ends. De novo telomere addition is a deleterious event, which is effectively inhibited by the nuclear Pif1 (nPif1) helicase phosphorylated at the TLSSAES motif in response to DNA damage. In this study, it is reported that the same regulatory motif of nPif1 is also required for DSB repair via BIR. The requirement of the nPif1 TLSSAES sequence in BIR is dependent on the functional DNA damage response (DDR). Thus, nPif1 phosphorylation by the DDR machinery might mediate the role of nPif1 in BIR. In contrast, the nPif1 regulatory motif is not essential for BIR at telomeres in cells lacking telomerase. These observations indicate that the mechanism of nPif1 function in DSB repair via BIR and in BIR at telomeres might be different. In this work, a protocol for nPif1 pull-down was optimized to reveal the mechanism of the phosphorylation-dependent nPif1 functions in cells undergoing DNA repair, i. e. the mechanism of nPif1-mediated inhibition of de novo telomere addition and promoting DSB repair via BIR. In future, this protocol can be used to dissect the role of nPif1 in DNA repair through the identification of its potential interacting partners. The Srs2 helicase negatively regulates HR via dismantling Rad51 filaments. According to preliminary data from the laboratory of Sveta Makovets, Srs2 also promotes de novo telomere addition at DSBs in a Rad51-dependent manner. The work presented here establishes that Srs2 is dispensable for telomerase-mediated addition of TG1-3 repeats to DSBs. Instead, Srs2 is required for the reconstitution of the complementary DNA strand after telomerase action, thus ensuring the completion of de novo telomere addition. Overall, this study demonstrates that recombination-dependent DSB repair and de novo telomere addition share common regulatory components, i. e. the nPif1 helicase phosphorylated in response to DNA damage and the Srs2 helicase. Phosphorylated nPif1 promotes DSB repair via BIR in addition to its known role in inhibition of telomerase at DSBs, whereas Srs2 uses its well established ability to remove Rad51 from ssDNA to promote the restoration of dsDNA and thus to complete de novo telomere addition.

Maintaining genomic integrity is crucial for cell survival. In Escherichia coli, Rec-Amediated homologous recombination (HR) plays an essential role in the repair of DNA double-strand breaks (DSB) and the SOS response through a series of highly dynamic interactions with the chromosome. A greater understanding of the mechanism of homologous recombination requires quantitative analysis of genomic studies in live cells. The aim of this thesis was to investigate the dynamics of the RecA-DNA interactions in vivo following the induction of a site-specific DSB in the chromosome of E. coli. This DSB is caused by the cleavage of a DNA hairpin by the hairpin-specific endonuclease, SbcCD. The DNA hairpin is formed only on the lagging strand template of replication by a 246 bp-interrupted palindrome. As a result cleavage only occurs on one sister chromosome, leaving one unbroken chromosome to serve as a template for repair by HR. Here, this system has been used as a basis to develop a method that combines chromatin immunoprecipitation with quantitative PCR (ChIP-qPCR) and next-generation sequencing (ChIP-Seq) to quantify RecA protein binding during the active repair of a single chromosomal DSB. This study reports that DSB-dependent RecA binding is stimulated in response to the eight base DNA sequence Chi (5’-GCTGGTGG-3’). Increasing the number of Chi sites close to the DSB stimulates more RecA loading to DNA, with ChIP-Seq analysis also revealing a role for subsequent Chi sites in RecA binding during DSBR. If the Chi sites close to the DSB are removed then Chi-dependent RecA binding to DNA can be observed at distances greater than 100 kb from the DSB, suggesting that these subsequent Chi sites can be engaged in DSBR. Through collaboration, these in vivo data were combined with stochastic modeling to determine that, in vivo, Chi is recognised by the RecBCD complex with an efficiency of 20- 35%. The genomic analysis also revealed two unexpected aspects of RecA protein binding. First, ChIP-Seq analyses identified that following a DSB at lacZ there is RecA enrichment detected in the terminus region of the E. coli chromosome. This RecA binding is Chi-dependent, indicating a role for HR. Second, DSB-independent binding was observed at the RNA encoding genes dispersed throughout the chromosome. A temporal analysis of RecA dynamics was also performed. These analyses revealed that RecA binding to DNA near the DSB is extremely dynamic, cycling between periods of high RecA enrichment and periods of low RecA enrichment. This is the first in vivo study of DSB-dependent RecA-DNA distribution and dynamics in recombination proficient E. coli cells.