Every living organism is programmed to reproduce and to pass genetic information to descendants. The information has to be carefully copied and accurately transferred to the next generation.  Therefore organisms have developed the network of conserved mechanisms to survey the protection and precise transfer of the genetic information. Such mechanisms are called checkpoints and they monitor the correct execution of different cell programs. The DNA damage and the replication blocks are surveyed by the conserved Mec1-Rad53 (human ATM/ATR and Chk2, respectively) protein kinase cascade. Mec1 and Rad53 are essential for survival and when activated orchestrate the multiple cellular responses, including the activation of the ribonucleotide reductase (RNR), to the genotoxic stress. RNR is an enzyme producing all four dNTPs - the building blocks of the DNA - and is instrumental for the maintenance both proper concentration and balance of each of dNTPs. The appropriate concentration of the dNTPs should be strictly regulated since inadequate dNTP production can impede many cellular processes and lead to higher mutation rates and genome instability. Hence RNR activity is regulated at many levels, including allosteric and transcriptional regulation and the inhibition at protein level. In our research, we addressed the question of the transcriptional regulation of RNR and the consequences of dNTP malproduction in the terms of the genomic stability. In yeast S. cerevisiae, four genes encode RNR: 2 genes encode a large subunit (RNR1 and RNR3) and 2 genes encode a small subunit (RNR2 and RNR4). All 4 genes are DNA-damage inducible: transcription of RNR2, RNR3 and RNR4 is regulated via Mec1-Rad53-Dun1 pathway by targeting the transcriptional repressor Crt1 (Rfx1) for degradation; on the contrary, RNR1 gene promoter does not contain Crt1-binding sites and is not regulated through the Mec1-Rad53-Dun1 pathway. Instead, we show that intrastrand cross (X)-link recognition protein (Ixr1) is required for the proper transcription of the RNR1 gene and maintenance of the dNTP pools both during unperturbed cell cycle and after the DNA damage. Thus, we identify the novel regulator of the RNR1 transcription. Next, we show that the depletion of dNTP pools negatively affects genome stability in the hypomorphic mec1 mutants: the hyper-recombination phenotype in those mutants correlates with low dNTP levels. By introducing even lower dNTP levels the hyper-recombination increased even further and conversely all the hyper-recombination phenotypes were suppressed by artificial elevation of dNTP levels. In conclusion, we present Ixr1 as a novel regulator of the RNR activity and provide the evidence of role of dNTP concentration in the genome stability.

<p>The complete and faithful duplication of the genome is essential to ensure normal cell division and organismal development. Eukaryotic DNA replication is initiated at multiple sites termed origins of replication that are activated at different time through S phase. The replication timing program is regulated by the S-phase checkpoint, which signals and repairs replicative stress. Eukaryotic DNA is packaged with histones into chromatin, thus DNA-templated processes including replication are modulated by the local chromatin environment such as post-translational modifications (PTMs) of histones.</p><p>One such epigenetic mark, methylation of lysine 20 on histone H4 (H4K20), has been linked to chromatin compaction, transcription, DNA repair and DNA replication. H4K20 can be mono-, di- and tri-methylated. Monomethylation of H4K20 (H4K20me1) is mediated by the cell cycle-regulated histone methyltransferase PR-Set7 and subsequent di-/tri- methylation is catalyzed by Suv4-20. Prior studies have shown that PR-Set7 depletion in mammalian cells results in defective S phase progression and the accumulation of DNA damage, which may be partially attributed to defects in origin selection and activation. Meanwhile, overexpression of mammalian PR-Set7 recruits components of pre-Replication Complex (pre-RC) onto chromatin and licenses replication origins for re-replication. However, these studies were limited to only a handful of mammalian origins, and it remains unclear how PR-Set7 impacts the replication program on a genomic scale. Finally, the methylation substrates of PR-Set7 include both histone (H4K20) and non-histone targets, therefore it is necessary to directly test the role of H4K20 methylation in PR-Set7 regulated phenotypes. </p><p>I employed genetic, cytological, and genomic approaches to better understand the role of H4K20 methylation in regulating DNA replication and genome stability in Drosophila melanogaster cells. Depletion of Drosophila PR-Set7 by RNAi in cultured Kc167 cells led to an ATR-dependent cell cycle arrest with near 4N DNA content and the accumulation of DNA damage, indicating a defect in completing S phase. The cells were arrested at the second S phase following PR-Set7 downregulation, suggesting that it was an epigenetic effect that coupled to the dilution of histone modification over multiple cell cycles. To directly test the role of H4K20 methylation in regulating genome integrity, I collaborated with the Duronio Lab and observed spontaneous DNA damage on the imaginal wing discs of third instar mutant larvae that had an alanine substitution on H4K20 (H4K20A) thus unable to be methylated, confirming that H4K20 is a bona fide target of PR-Set7 in maintaining genome integrity. </p><p>One possible source of DNA damage due to loss of PR-Set7 is reduced origin activity. I used BrdU-seq to profile the genome-wide origin activation pattern. However, I found that deregulation of H4K20 methylation states by manipulating the H4K20 methyltransferases PR-Set7 and Suv4-20 had no impact on origin activation throughout the genome. I then mapped the genomic distribution of DNA damage upon PR-Set7 depletion. Surprisingly, ChIP-seq of the DNA damage marker γ-H2A.v located the DNA damage to late replicating euchromatic regions of the Drosophila genome, and the strength of γ-H2A.v signal was uniformly distributed and spanned the entire late replication domain, implying stochastic replication fork collapse within late replicating regions. Together these data suggest that PR-Set7-mediated monomethylation of H4K20 is critical for maintaining the genomic integrity of late replicating domains, presumably via stabilization of late replicating forks.</p><p>In addition to investigating the function of H4K20me, I also used immunofluorescence to characterize the cell cycle regulated chromatin loading of Mcm2-7 complex, the DNA helicase that licenses replication origins, using H4K20me1 level as a proxy for cell cycle stages. In parallel with chromatin spindown data by Powell et al. (Powell et al. 2015), we showed a continuous loading of Mcm2-7 during G1 and a progressive removal from chromatin through S phase.</p> / Dissertation

Telomeres are the ends of linear chromosomes tasked with preventing their recognition by the DNA damage machinery and providing a mechanism to solve the end replication problem. The telomeric DNA is mostly double-stranded, but it terminates in a 3’ protrusion termed the G-overhang. Telomeres utilize telomerase, a reverse transcriptase, to elongate the telomere, and thus, solve the end replication problem. Both the double strand region and the G-overhang are bound by specific proteins to facilitate the objectives of the telomere. First discovered in budding yeast, the CST (Cdc13(CTC1)/Stn1/Ten1) complex binds to the G-overhang and is important for both chromosome end protection and telomere replication. Work reported in this dissertation provided the first evidence that CST was present outside of yeast, which led to its subsequent identification in a number of vertebrates. Here I present the identification and characterization of the three components of CST in Arabidopsis thaliana. Similar to yeast, Arabidopsis CST is required for telomere length maintenance, for preventing telomere recombination and chromosome end-to-end fusions. Mutations in the CST complex result in severe genomic instability and stem cells defects. My research also shows that CST and telomerase act synergistically to maintain telomere length. Together these data provide evidence for an essential role for CST in maintaining telomere integrity. Unexpectedly, I discovered that the TEN1 component of CST may have a more complex role than other members of the heterotrimer. The majority of telomere-related functions we can assay using molecular and cytological approaches are shared by CTC1, STN1 and TEN1, though TEN1 has additional roles in maintaining genome stability, modulating telomerase activity and possibly non-telomeric functions in the chloroplast. I also present genetic evidence that TEN1 and STN1 act in the same pathway for the maintenance of telomere length and chromosome end protection. Interestingly, however, disrupting the STN1/TEN1 interaction reveals a separation of STN1 function for chromosome end protection versus telomere length maintenance. Finally, I describe the design and creation of a library of STN1 and TEN1 mutants that will be used to further characterize their functions and their interaction partners. By disrupting such interactions, it will be possible to elucidate the functional significance of these interactions, and thus, provide new insight into how CST functions in Arabidopsis.

The genome of each cell is under constant threat from various forms of DNA damage. In order to protect themselves from this danger, cells possess a number of pathways able to resolve DNA lesions. The addition of poly(ADPribose) is a post-translational modification produced by attaching successive ADP-ribose moieties to a protein acceptor, forming long chains. Enzymes called poly(ADP-ribose) polymerases (PARPs) catalyse the production of these modifications, and a number of different PARPs have been linked to the process of DNA repair, including PARP1, PARP2 and PARP3. How these enzymes might function together to facilitate the repair of different lesions is unclear. Furthermore, inhibitors that target these enzymes are in clinical use for their ability to kill homologous recombination deficient tumour cells, through a mechanism of synthetic lethality. Which subset of PARPs is necessary to inhibit to achieve maximum efficacy of these agents has not been assessed. I use genome editing to generate cells disrupted for these PARPs in different combinations. Whilst loss of PARP1 compromises cellular tolerance to homologous recombination deficiency, this is independent of the status of PARP2 and PARP3, indicating the development of PARP1-specific inhibitors may hold therapeutic potential. In contrast to these observations, I uncover strong redundancy between PARP1 and PARP2 in the repair of damaged DNA bases through the base excision repair (BER) pathway. I also identify BER independent roles of both PARP1 and PARP2 in resolving replication forks that have collided with BER-intermediates, through promoting the stability of Rad51 nucleofilaments via an Fbh1-dependent mechanism. Thus PARP1 and PARP2 perform two closely-linked functions in response to cellular base damage promoting resolution of these lesions directly through BER, and stabilising replication forks which have encountered BER intermediates.

No description available.

No description available.

Genome stability has great importance in maintaining cell viability and optimal functionality of cellular processes. Loss of genome stability can lead to cell death in the simplest organisms and to deregulation of the cell proliferation machinery in higher organisms, potentially causing cancer or morbid states. The Single Stranded DNA Binding (SSB) protein of Escherichia coli is an essential protein that binds and stabilises ssDNA stretches. Its role is particularly crucial during DNA replication, recombination and repair processes and it has therefore been predicted to play a prominent role in the maintenance of genome stability. The role of SSB in genome instability was investigated using an E. coli strain in which, the expression of the ssb gene was placed under the control of the arabinose promoter. The level of SSB protein present in the cell could therefore be tuned by varying the arabinose concentration in the medium. A wide characterisation of the behaviour of the strain at low SSB level was carried out. Viability and growth tests showed that a threshold level of protein is required to allow normal growth. Microscopy analyses were carried out to follow cell division, nucleoid morphology and SOS response activation. Cells grown at low SSB level, showed a phenotype consistent with impaired cell division and altered nucleoid morphology. The SOS response was activated at low SSB levels and cell elongation was detected. Lowering the arabinose concentration in solid medium allowed the selection of suppressor strains that could form colonies under the new conditions. Sequencing of the entire genome of one such suppressor strain was carried out revealing a possible candidate for the phenotype change. The stability of a 105bp and of a 246bp DNA imperfect palindromes and the stability of CAG·CTG trinucleotide repeat arrays, inserted in the E. coli chromosome, were investigated in correlation to the SSB cellular level. Lowering the SSB level in cells grown on solid medium, increased the instability of the 105bp palindrome presumably by increasing the number of slippage events. On the other hand, SSB overexpression did not have an effect on the stability of the 246bp palindrome. The stability of a leading strand (CAG)75 repeat array was highly increased by overexpressing SSB, while the same effect was not observed for a leading strand (CTG)137 repeat array. Furthermore, excess SSB caused a change in the deletion size distribution profile for the leading strand (CAG)75 strain, lowering the bias towards big deletions. This is consistent with SSB being able to preferentially impede the formation of big DNA hairpins. Also, SbcCD nuclease was shown to have an effect on the deletion size distribution profile of the leading strand (CTG)137 strain. The lack of SbcCD led to a slight reduction of the number of big deletions.

Obstacles such as DNA damage can block the progression of DNA replication forks. This is a major source of genome instability that can lead to cell transformation or death. The budding yeast MMS1 and MMS22 genes were identified in a screen for mutants that were hypersensitive to DNA alkylation that blocks replisome progression. I set out to investigate the cellular roles of these genes and found that cells lacking MMS1 or MMS22 are hypersensitive to a wide variety of genotoxins that stall or block replication forks, and are severely defective in their ability to recover from DNA alkylation damage. Homologous recombination (HR) is an important mechanism for the rescue of stalled or blocked replication forks and for the repair of double-strand breaks (DSBs). Strikingly, MMS1 and MMS22 are required for HR induced by replication stress but not by DSBs, and the underlying mechanisms were explored.I next identified the uncharacterized protein C6ORF167 (MMS22L) as a putative human Mms22 orthologue. MMS22L interacts with NF?BIL2/TONSL, the histone chaperone ASF1 and subunits of the MCM replicative helicase. MMS22L colocalizes with TONSL at perturbed replication forks and at sites of DNA damage. MMS22L and TONSL are important for the repair of collapsed replication forks as depletion of MMS22L or TONSL from human cells causes DNA damage during S–phase and hypersensitivity to agents that cause fork collapse. These defects are consistent with the observations that MMS22L and TONSL are required for the efficient loading of the RAD51 recombinase onto resected DNA ends and for efficient HR. These data indicate that MMS22L and TONSL are novel regulators of genome stability that enable efficient HR.

The mismatch repair system (MMR) is an important pathway for maintaining genome stability because it can remove the errors generated while the cell is replicating. If these errors are left uncorrected, they can lead to genomic mutations. Thus, the deficient mismatch repair system is associated with the development of sporadic cancers and degenerative diseases such as Lynch Syndrome. MMR involves a set of proteins including MutSα, MutLα, MutLβ, and MutLγ. MutSα and MutLα play a major role in MMR whereas MutLβ and MutLγ provide minor contributions to this pathway. Recent studies have suggested that MutLβ and MutLγ are involved in the triplet DNA repeat expansion pathway. Our study in Saccharomyces cerevisiae shows important preliminary data of Mlh3 in maintaining genomic stability. We studied its interactions with other proteins involved in the mismatch repair system. Interestingly, Mlh3 interactions with Msh3 and Pol 35 DV suggest that there is a predilection of MutSβ and MutLγ to work in the lagging strand. Additionally, Top1, Mlh3and Msh3our results have shown that these genetic interactions could lead to an increase in mistakes in the MMR pathway. Moreover, it could lead to the suggestion that they are also involved in another pathway such as transcription. Finally, we confirm the involvement of Mlh3 in the resolution of frameshift mutations in the His-7A locus. Even though they are interesting results is too early to make final conclusions. Further analysis needs to be done.

The final stages of replication fork termination requires the timely and orderly orchestration of catalytic and enzymatic activities. Given the complexity of this process, it is conceivable that the final stages of fork termination is susceptible to problems that could trigger recombination, which could lead to deleterious genomic rearrangements if ectopic homologous sequences are recombined. Using the site-specific RTS1 barrier in fission yeast, I demonstrated that fork termination is generally not a recombinogenic process, and that hyper-recombination-induced by fork blockage at RTS1 is largely a result of replication fork restart. To investigate the actual mechanisms and proteins, which drive and influence recombination at a replication barrier, I studied the MHF proteins, which assist Fml1 in limiting crossovers during double-strand break (DSB) repair and promoting Rad51-mediated recombination at impeded replication forks, and are also components of the constitutive centromere-associated network (CCAN). Intriguingly, structural studies revealed that the MHF can exist as an octamer in vitro. I examined the biological significance of octameric MHF by employing three mutations that disrupt the octamer configuration in vitro. In fission yeast, these mutations cause hypersensitivity to methyl methanesulfonate (MMS), suggesting that the MHF octamer may have a role in DNA repair. One of the “octamerisation” mutants, exhibits greater hypersensitivity to MMS than the other two, and biochemical experiments indicated that this is because it confers an additional defect in MHF’s interaction with Fml1. Further genetic experiments on this mutant suggest that the ability of Fml1 to unwind D-loops depends more critically on its interaction with MHF than fork reversal. Additionally, I showed a synergistic interaction between Dcr1 and MHF, and demonstrated that in the absence of Dcr1, there is a greater need for recombination to tolerate/repair DNA damage. Lastly, I uncovered a novel function for the MHF in controlling the initiation of septation.