Genome instability is a hallmark of human cancers. Patients with Bloom’s syndrome, a rare chromosome breakage syndrome caused by inactivation of the RecQ helicase BLM, result in phenotypes associated with accelerated aging and develop cancer at a very young age. Patients with Bloom’s syndrome exhibit hyper-recombination, but the role of BLM and increased genomic instability is not fully characterized. Sgs1, the only member of the RecQ family of DNA helicases in Saccharomyces cerevisiae, is known to act both in early and late stages of homology-dependent repair of DNA damage. Exo1, a 5′–3′ exonuclease, first discovered to play a role in mismatch repair has been shown to participate in parallel to Sgs1 in processing the ends of DNA double-strand breaks, an early step of homology-mediated repair. Here we have characterized the genetic interaction of SGS1 and EXO1 with other repair factors in homology-mediated repair as well as DNA damage checkpoints, and characterize the role of post-translational modifications, and protein-protein interactions in regulating their function in response to DNA damage. In S. cerevisiae cells lacking Sgs1, spontaneous translocations arise by homologous recombination in small regions of homology between three non-allelic, but related sequences in the genes CAN1, LYP1, and ALP1. We have found that these translocation events are inhibited if cells lack Mec1/ATR kinase while Tel1/ATM acts as a suppressor, and that they are dependent on Rad59, a protein known to function as one of two sub-pathways of Rad52 homology-directed repair. Through a candidate screen of other DNA metabolic factors, we identified Exo1 as a strong suppressor of chromosomal rearrangements in the sgs1∆ mutant. The Exo1 enzymatic domain is located in the N-terminus while the C-terminus harbors mismatch repair protein binding sites as well as phosphorylation sites known to modulate its enzymatic function at uncapped telomeres. We have determined that the C-terminus is dispensable for Exo1’s roles in resistance to DNA-damaging agents and suppressing mutations and chromosomal rearrangements. Exo1 has been identified as a component of the error-free DNA damage tolerance pathway of template switching. Exo1 promotes template switching by extending the single strand gap behind stalled replication forks. Here, we show that the dysregulation of the phosphorylation of the C-terminus of Exo1 is detrimental in cells under replication stress whereas loss of Exo1 suppresses under the same conditions, suggesting that Exo1 function is tightly regulated by both phosphorylation and dephosphorylation and is important in properly modulating the DNA damage response at stalled forks. It has previously been shown that the strand exchange factor Rad51 binds to the C-terminus of Sgs1 although the significance of this physical interaction has yet to be determined. To elucidate the function of the physical interaction of Sgs1 and Rad51, we have generated a separation of function allele of SGS1 with a single amino acid change (sgs1-FD) that ablates the physical interaction with Rad51. Alone, the loss of the interaction of Sgs1 and Rad51 in our sgs1-FD mutant did not cause any of the defects in response to DNA damaging agents or genome rearrangements that are observed in the sgs1 deletion mutant. However, when we assessed the sgs1-FD mutant in combination with the loss of Sae2, Mre11, Exo1, Srs2, Rrm3, and Pol32 we observed genetic interactions that distinguish the sgs1-FD mutant from the sgs1∆mutant. Negative and positive genetic interactions with SAE2, MRE11, EXO1, SRS2, RRM3, and POL32 suggest the role of the physical interaction of Sgs1 and Rad51 is in promoting homology-mediated repair possibly by competing with single-strand binding protein RPA for single-stranded DNA to promote Rad51 filament formation. Together, these studies characterize additional roles for domains of Sgs1 and Exo1 that are not entirely understood as well as their roles in combination with DNA damage checkpoints, and repair pathways that are necessary for maintaining genome stability.

No description available.

The pathogen Francisella tularensis subsp. tularensis has been classified as a Center for Disease Control (CDC) select agent. However, little is still known of what makes the bacteria cause dis-ease, especially the highly virulent type A1 strains. The work in this dissertation focused on type A1 strains from the Inzana laboratory, including a wildtype virulent strain TI0902, an avirulent chemical mutant strain TIGB03 with a Single Nucleotide Polymorphism in the wbtK gene, and several complemented mutants, [wbtK+]TIGB03, with dramatic differences in virulence and growth rates. One of the complemented clones (Clone12 or avp-[wbtK+]TIGB03-C12) was aviru-lent, but protected mice against challenge of a lethal dose of TI0902 and was considered as a possible vaccine strain. Whole genome sequencing was performed to identify genetic differences between the virulent, avirulent and protective strains using both Roche/454 and Illumina next-generation sequencing technologies. Additionally, RNASeq analysis was performed to identify differentially expressed genes between the different strains. This comprehensive genomic analysis revealed the critical role of transposable elements in inducing genomic instability resulting in large du-plications and deletions in the genomes of the chemical mutant and the complemented clones that in turn affect gene dosage and expression of genes known to regulate virulence. For exam-ple, whole genome sequencing of the avirulent chemical mutant TIGB03 revealed a large 75.5 kb tandem duplication flanked by transposable elements, while the genome of a virulent Clone01 (vir-[wbtK+]TIGB03-C1) lost one copy of the 75.5 kb tandem duplicated region but gained a tandem duplication of another large 80kb region that contains a virulence associated transcription factor SspA. RNAseq data showed that the dosage effect of this extra region in Clone1 suppresses expression of MglA regulated genes. Since MglA regulates genes that are known to be crucial for virulence, including the well-studied Francisella Pathogenicity Island (FPI), these results suggest that gene dosage effects arising from large duplications can trigger unknown virulence mechanisms in F. tularensis subsp. tularensis. These results are important especially when designing live vaccine strains that have repeated insertion elements in their genomes. / Ph. D.

Acquisition of invasiveness through extracellular matrix is a crucial characteristic of transition to malignancy in the breast. It was previously shown that Polo-like kinase 1 (PLK-1), a mitotic kinase and genome stability regulator, is involved in acquisition of invasiveness in a breast cell model (HMT-3522 cell line) of pre-invasive to invasive transition. This and other data led to the suggestion that a new class of genes called GISEM for Genome Instability and Extracellular Matrix Invasiveness may exist. Previous lab data show that XRCC3 is found downregulated in progression from preinvasive to invasive phenotype. This led to the hypothesis that XRCC3 may be a negative regulator of invasion. To support this hypothesis, overexpression of XRCC3 in the invasive T4-2 cells downregulated invasion, but also growth. In order to verify the role of XRCC3 in invasiveness, and determine whether it is independent from any effects on growth, we tested the effect of downregulating XRCC3 on the invasiveness of T4-2 cells. Short-term downregulation of XRCC3 using siRNAs produced a significant increase in invasiveness, suggesting a role for XRCC3 as a negative regulator of invasion. During the invasion assay time course, XRCC3 downregulation had no effect on growth or apoptosis supporting the idea that this is a direct effect on invasion and not an artifact of the assay. XRCC3 is one amongst the five members of the RAD51 paralog family, consisting of accessory proteins or RAD51 cofactors (namely RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) which interact with each other to form complexes (BCDX2, BC, DX2 and CX3) that collaboratively assist RAD51 in homologous recombinational repair (HRR) of DNA double-strand breaks. To see if these interactions are important in terms of invasion, as they have been demonstrated for DNA repair, we studied the effect of XRCC3 downregulation on the levels of RAD51 paralogs. We found lowered levels of RAD51C, but not RAD51B or RAD51D, when XRCC3 was downregulated. Since XRCC3 forms the CX3 complex with RAD51C, we downregulated RAD51C using siRNAs in T4-2 cells and found this to significantly increase invasiveness. Consistent with previous findings by other groups, downregulating RAD51C also lead to decreased levels of XRCC3 in invasive T4-2 cells. These results suggest that the XRCC3-RAD51C interaction is important for invasion as well as the previously studied DNA repair function. In delineating the mechanism by which XRCC3 acts as a negative regulator of invasion, we further questioned if XRCC3 alters secreted factors that are important for the invasiveness of T4-2 cells and tested the effects of conditioned medium (CM) from XRCC3 altered T4-2 cells on parental T4-2 cells’ ability to invade. Results show a significant increase in invading T4-2 cells when suspended in CM from XRCC3 siRNA transfected T4-2 cells, suggesting a direct effect of XRCC3 siRNAs on the ability of T4-2 CM to induce invasiveness in T4-2 cells. Furthermore, we investigated the effects of XRCC3 inhibition on cell surface integrins and focal adhesion kinase (FAK). Indirect immunofluorescence results show increased formation of focal adhesions containing two phosphorylated FAK residues- autophosphorylated FAK-Y397 and FAK-Y861 (previously implicated in increased migration and invasion of tumor cells) in XRCC3 siRNA transfected T4-2 cells. Overall, these results support a new role of XRCC3 in invasion, in addition to its previously reported role in DNA repair. These findings imply that loss of XRCC3 function in cancer progression would upregulate invasion as well as downregulate DNA repair and genome stability. Therefore, stabilization of XRCC3 function could provide a promising therapeutic against breast cancer progression. The dual role of XRCC3 in invasion and DNA repair also renders it an attractive candidate risk biomarker of breast pre-cancer to invasive cancer progression.

Jusqu'à présent, les modifications de la réplication de l'ADN entre cellules normales et cancéreuses ont été peu étudiées. Dans ce travail, nous avons utilisé le séquençage des fragments d'Okazaki, une technique récemment développée au laboratoire, pour déterminer la directionalité des fourches de réplication dans plusieurs lymphomes de Burkitt (LB), qui surexpriment l'oncoprotéine Myc à la suite de translocations chromosomiques spécifiques, ainsi que dans des lignées lymphoblastoides contrôles (LLC) et dans des léiomyosarcomes (LMS). Les profils de directionalité des fourches de réplication permettent de déduire la localisation et l'efficacité des sites d'initiation et de terminaison de la réplication le long du génome. Nous avons observé de nombreuses (~2000) différences de zones d'initiation entre les lignées Raji (LB) et GM06990 (LLC) ainsi qu'entre les lignées BL 79 et IARC385, une paire LB/LLC provenant d'un même patient. Nous avons détecté un nombre comparable de différences en comparant deux à deux les lignées étudiées. Cependant, les profils de BL79 et de Raji (deux LB) sont un peu plus proches l'un de l'autre que de la lignée contrôle GM06990. Ceci suggère l'existence de changements de la réplication récurrents dans les lignées LB. L'importance des différences observées entre les lignées IARC385 et GM06990 indique de façon surprenante une grande variabilité entre les LLC normales, provenant de différents individus. De façon intéressante, de nombreuses différences observées entre les lignées LB et LLC sont associées à des changements de l'expression des gènes ou de la liaison de l'oncoprotéine Myc. La comparaison des profils des deux LMS avec tous les profils disponibles au laboratoire montre que c'est à celui de fibroblastes normaux (IMR90) qu'ils ressemblent le plus. Ceci suggère que les cellules de tumeurs musculaires lisses auraient subi une transformation fibroblastique au cours de la tumorigénèse. Des données récentes suggèrent que les champs magnétiques peuvent perturber certains processus cellulaires comme l'assemblage du cytosquelette. Nous avons utilisé le séquençage de fragment d'Okazaki pour rechercher d'éventuels effets d'un champ magnétique sur la réplication de l'ADN chez la levure. Aucun effet du champ magnétique sur la directionalité des fourches de réplication n'a été détecté. / Changes in DNA replication profiles between cancer and normal cells have been poorly explored. In this work, sequencing of Okazaki fragments, a novel methodology developed in the laboratory, was used to determine replication fork directionality (RFD) in several Burkitt's lymphomas (BL), which overexpress the Myc oncoprotein due to specific chromosomal translocations, and control normal lymphoblastoid cell lines (LCL), and in leiomyosarcomas (LMC). RFD profiles allow to infer the location and efficiency of replication initiation and termination sites genome-wide. A larger number (~2000) of differences in replication initiation zones were observed genome-wide between Raji (BL) and GM06990 (LCL), and between BL79 and IAR385, a BL / LCL pair of cell lines established from a single patient. Comparably large numbers of changes were slightly more similar to each other than to GM06990. This suggests the occurrence of some recurrent replication changes in BL cell lines. The large number of changes observed between IARC385 and GM06990 also indicates an unexpectedly large variation between normal LCLs of different individuals. Interestingly, many changes in RFD profiles between BLs and and LCLs are associated with cell-type specific gene expression and differential binding of the Myc oncoprotein. Comparison of the two LMS profiles with all RFD profiles available in the laboratory reveals that they most resemble normal fibroblasts (IMR90). This suggests that the smooth muscle cancer cells might have undergone a fibroblastic transformation during tumorigenesis. Magnetic fields have been reported to perturb cellular processes such as cytoskeleton assembly. Sequencing of Okazaki fragments was used in a preliminary investigation of the possible effects of magnetic fields on DNA replication in yeast cells. No effect of magnetic fields on replication fork directionality were observed.

Les cellules sont constamment exposées à des agents endommageant de l'ADN d'origine exogène, notamment les rayons ultraviolets, les irradiations γ, et l'exposition aux agents chimiques génotoxiques, mais également d'origine endogène générés par le métabolisme cellulaire. De plus en plus d'évidences montrent que la transcription est un processus biologique qui peut mettre en péril l'intégrité du génome. Un mécanisme actuellement très étudié qui lie la transcription à l'instabilité génomique est la formation des boucles R (R-loops), des structures hybrides ARN:ADN qui exposent un ADN simple brin déplacé. Ces structures aberrantes se présentent en tant que sous-produits de la transcription et/ou lors de l'interférence entre la réplication et la transcription, et plus récemment ils ont été montrées s'accumuler lorsque la biogénèse de l'ARNm est perturbée. La persistance des boucles R est une source importante d'instabilité génomique car elle peut générer des cassures double brin de l'ADN et favoriser la recombinaison. Pour faire face aux conséquences néfastes des endommagements de l'ADN, les cellules activent une cascade élaborée de voies de signalisation qui permet de coordonner la prolifération cellulaire avec la réparation de l'ADN. L'ensemble de ces acteurs moléculaires constitue un réseau de réponse aux dommages de l'ADN qui est indispensable pour la stabilité génomique. Récemment chez la levure, l'activation transitoire de ce réseau a été également proposée être important dans la coordination de la transcription et de la réplication, afin d'éviter d'une part des contraintes topologiques et d'autre part la formation de structures aberrantes générées lors de conflits entre ces deux processus cellulaires essentiels. Dans la perspective d'identifier des nouveaux gènes impliqués dans ce réseau de signalisation, un crible fonctionnel in vitro précédemment établi au laboratoire a conduit à l'identification de Ddx19, une hélicase à motif DEAD-box, en tant que nouvel élément répondant à l'endommagement de l'ADN. Ddx19 interagit avec le pore nucléaire via CAN/Nup214, et il est impliqué dans l'export des ARNm grâce à son activité hélicase et ATPase, stimulé par les facteurs IP6 et Gle1. Le présent travail de thèse dévoile une nouvelle fonction de Ddx19 distincte de son rôle connu dans l'export de l'ARNm. Je pu montrer que, lors de l'induction des dommages à l'ADN par les rayons UV, Ddx19 se relocalise transitoirement de la face cytoplasmique du nucléopore vers le noyau de façon dépendant d'ATR. L'inactivation de Ddx19 entraîne des endommagements spontanées dépendant de la prolifération, démontré par l'activation de la voie de signalisation d'ATM-Chk2 et la formation de foyers nucléaires de γH2AX et 53BP1. Ces phénotypes sont concomitants avec le ralentissement des fourches de réplication qui ne peuvent plus redémarrer après leur blocage par la camptothécine. En outre, les cellules déplétées de Ddx19 présentent une forte accumulation des boucles R nucléaires, enrichi dans le compartiment nucléolaire, et aussi autour de la périphérie nucléaire. Par ailleurs, ces cellules présentent une viabilité réduite et une létalité synthétique lorsque la déplétion de Ddx19 est combinée avec l'inhibition de l'expression de la topoisomérase I. Je propose Ddx19 comme deuxième hélicase nécessaire pour la résolution des boucles R, et qui fonctionne à côté mais de façon indépendante de la Senataxin, l'hélicase précédemment connue pour résoudre ces structures in vivo chez les cellules de mammifères. Je démontre que cette nouvelle fonction de Ddx19 ne dépend pas de son interaction avec le pore nucléaire, mais plutôt de son activité hélicase et d'un résidu de sérine phosphorylée par Chk1 qui stimule sa relocalisation vers le noyau. Ces données proposent Ddx19 en tant que nouvelle ARN hélicase qui facilite la coordination de la réplication et la transcription, médiée par ATR à travers de la résolution des boucles R, préservant ainsi l'intégrité du génome. / Cells are continuously challenged by DNA damage resulting from external cues as UV light, γ-irradiation and exposure to genotoxic chemicals, as well as from endogenous stress caused by cellular metabolism. Growing evidence points to transcription as a biological process that could adversely affect genome integrity. One currently highly investigated mechanism by which transcription can induce genome instability is through the formation of R-loops, RNA:DNA hybrid structures exposing a displaced single-stranded DNA tract. These aberrant structures occur as byproducts of transcription and/or upon interference between replication and transcription, and more recently were also shown to accumulate upon disruption of mRNA biogenesis and processing. Persistent unresolved R-loops are a potent source of genomic instability as they ultimately generate double strand breaks and promote recombination events. To deal with the deleterious consequences of DNA damage, cells activate elaborate DNA damage response (DDR) pathways to delay cell division and stimulate repair of lesions, thus preserving genome stability. Recently in yeast transient DDR activation has also been proposed to be important in the coordination of transcription and replication, in order to avoid topological constraints and the formation of aberrant structures generated upon collision of their machineries. By means of an in vitro screen aimed at identifying new DDR genes, we isolated Ddx19, a DEAD-Box helicase known to be involved in mRNA export, as a novel DNA damage responsive gene. Ddx19 interacts with the nucleopore complex via nucleoporin CAN/Nup214, and is involved in mRNA remodelling and export through its ATPase and helicase activities, stimulated by IP6 and the Gle1 factor. My present thesis work unravels a novel function of Ddx19 in preserving genome stability in mammalian cells, distinct from its known role in mRNA export. I show that upon UV-induced damage, Ddx19 transiently relocalizes from the cytoplasmic face of the nucleopore to the nucleus in an ATR-dependent manner. Downregulation of Ddx19 gives rise to spontaneous, proliferation-dependent DNA damage, as determined by the specific activation of the ATM-Chk2 pathway and formation of γH2AX and 53BP1 nuclear foci. This is concomitant with the slowing down of replication forks that are unable to restart after being stalled with camptothecin. In addition, cells depleted of Ddx19 display strong accumulation of nuclear R-loops, enriched in the nucleolar compartment, and around the nuclear periphery. Moreover, these cells show low viability and exhibited synthetic lethality when combined with inhibition of topoisomerase I expression. I propose Ddx19 as a second helicase required for R-loops resolution, functioning alongside but independently of Senataxin, the first known RNA helicase to resolve these structures in vivo in mammalian cells. I provide evidence that this new function of Ddx19 does not depend on its interaction with the nuclear pore, but rather on its helicase activity and on a serine residue phosphorylated by Chk1 which promotes its relocalization into the nucleus upon damage. These data put forward Ddx19 as a novel RNA helicase that facilitates ATR-dependent coordination of DNA replication and transcription through R-loops resolution, thus preserving genome integrity.

A common feature of progeria syndromes is a premature aging phenotype and an enhanced accumulation of DNA damage arising from a compromised repair system. HGPS (Hutchinson-Gilford progeria syndrome) is a severe form of progeria in which patients accumulate progerin, a mutant lamin A protein derived from a splicing variant of the lamin A/C gene (LMNA). Progerin causes chromatin perturbations which result in the formation of DSBs (double-strand breaks) and abnormal DDR (DNA-damage response). In the present article, we review recent findings which resolve some mechanistic details of how progerin may disrupt DDR pathways in HGPS cells. We propose that progerin accumulation results in disruption of functions of some replication and repair factors, causing the mislocalization of XPA (xeroderma pigmentosum group A) protein to the replication forks, replication fork stalling and, subsequently, DNA DSBs. The binding of XPA to the stalled forks excludes normal binding by repair proteins, leading to DSB accumulation, which activates ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) checkpoints, and arresting cell-cycle progression.

La surexpression de la lamine B1, un composant majeur de l'enveloppe nucléaire, a été rapportée dans diverses tumeurs. Cependant, les causes et les conséquences de cette augmentation sur la stabilité du génome n'ont pas été étudiées à ce jour. En effet, l'instabilité du génome est considérée comme une caractéristique majeure des cellules cancéreuses. Pour assurer le maintien de la stabilité du génome, les cellules ont développé de multiples et complexes mécanismes parmi lesquels les voies de réparation de l'ADN et la gestion du stress réplicatif sont essentielles. Au cours de ma thèse, l'impact de l'augmentation de niveau de lamine B1 sur la stabilité du génome, en particulier sur la réparation de cassure double-brin (CDB) et sur le contrôle du stress réplicatif a été étudié. En effet, nous montrons qu'une augmentation de la lamine B1 entraîne une accumulation de CDB et leur persistance en réponse à l'irradiation (foyers γH2AX), en plus d'une sensibilité accrue à l'irradiation (formation de colonies et cassures chromosomiques). Les cellules surexprimant la lamine B1 montrent également des défauts de recrutement de 53BP1 aux sites de dommages d’ADN, couplés à une diminution de l'efficacité de la réparation de CDB par NHEJ (Non-Homologous End-Joining). De plus, nous avons identifié une interaction directe entre la lamine B1 et 53BP1 régulant le recrutement de ce dernier aux CDB. Nos résultats supportent un modèle dans lequel l'augmentation de la lamine B1 conduit à la séquestration de 53BP1, modifiant ainsi son recrutement aux CDBs. En parallèle, nous montrons que les cellules surexprimant la lamine B1 présentent des signes accrus de stress réplicatif tels que l'accumulation de foyers spontanés de p-RPA, l'augmentation des figures radiales lors du traitement par mitomycine C, et une sensibilité accrue au traitement par camptothécine. Nous avons en outre cherché à identifier les causes de l'augmentation du stress réplicatif dans ces cellules, et les conséquences potentielles, en particulier sur l'induction de phénotypes inflammatoires. En fait, nous montrons que la surexpression de la lamine B1 conduit à une diminution de l'efficacité de la réparation de CDB par la recombinaison homologue, couplée à un défaut de formation de foyers BRCA1 après irradiation. De plus, nous avons obtenu des données préliminaires suggérant une induction de l'inflammation lors de la surexpression de la lamine B1. En résumé, ce travail de Thèse a permis d’identifier un nouveau mécanisme régulant le recrutement de 53BP1 aux CDB par son interaction avec la lamine B1, et souligne le rôle de l'augmentation de la lamine B1 dans la promotion de l'instabilité génomique au moins partiellement par des défauts de réparation de CDB et une augmentation de stress réplicatif. Après confirmation de l'induction de phénotypes inflammatoires, nous aurions identifié des rôles de l'augmentation de la lamine B1 dans la promotion de deux caractéristiques majeures du cancer - l'instabilité génomique et l'inflammation - favorisant ainsi le rôle de la lamine B1 dans le développement tumoral et proposant cette dernière comme une cible thérapeutique antitumorale potentielle. / The overexpression of lamin B1, a major component of nuclear envelope, has been reported in various tumors. However, the causes and consequences of this increase on the genome stability have not been studied to date. Indeed, genome instability is considered a major hallmark of cancer cells. To ensure the maintenance of genome stability, cells have developed multiple complex mechanisms among which pathways of DNA repair and replication stress management are essential. Therefore, during my thesis the impact of an increased lamin B1 level on genome stability, in particular on double-strand break (DSB) repair and on the control of replication stress was studied. Indeed, we show that increased lamin B1 leads to an accumulation of DSBs and their persistence in response to irradiation (γH2AX foci), in addition to an increased sensitivity to irradiation (colony formation and chromosomal breaks). Lamin B1 overexpressing cells also show defects in the recruitment of 53BP1 to damage sites, coupled to a decreased efficiency of DSB repair by Non-Homologous End-Joining. Moreover, we identified a direct interaction between lamin B1 and 53BP1 regulating the latter’s recruitment to DSBs. Our results support a model where increased lamin B1 leads to the sequestration of 53BP1, thereby altering its recruitment to DSBs. In parallel, we show that cells overexpressing lamin B1 display increased signs of replication stress such as accumulation of spontaneous p-RPA foci, increased radial chromosomes upon mitomycin C treatment, and enhanced sensitivity to treatment with camptothecin. We further aimed to identify the causes of the increased replication stress in these cells, in addition to the potential consequences, in particular on the induction of inflammatory phenotypes. In fact, we show that lamin B1 overexpression leads to a decreased efficiency of DSB repair by Homologous Recombination, coupled to a defect in irradiation-induced BRCA1 foci formation. In addition, we obtained preliminary data suggesting a possible induction of inflammation upon lamin B1 overexpression. Altogether, this work identifies a novel mechanism regulating the recruitment of 53BP1 to damage sites through its interaction with lamin B1, and highlights the role of increased lamin B1 in promoting genome instability at least partially through defective DSB repair and increased replication stress. Upon confirming the induction of inflammatory phenotypes, we would have identified roles of increased lamin B1 in promoting two major hallmarks of cancer – genomic instability and inflammation - thereby favorizing a role for lamin B1 in tumor development and proposing the latter as a potential anti-tumor therapeutic target.

No description available.

DNA double-strand breaks (DSBs) are highly genotoxic lesions, which can promote chromosomal rearrangements and tumorigenesis through oncogene activation or loss of heterozygosity (LOH) at tumour suppressor loci. To identify new genes involved in DSB repair and genome stability, an S. pombe deletion library was screened for mutants which exhibited sensitivity to the DNA damaging agents bleomycin and/or MMS. 192 mutants were isolated which exhibited increased sensitivity to one or both of these agents. These mutants were further analysed in a sectoring assay and mutants sought which exhibited elevated levels of break-induced loss and rearrangement of a non-essential minichromosome. Using this approach 57 genes were identified, including all known homologous recombination (HR) and DNA damage checkpoint genes present in the library. Further, quantitative analysis of DSB repair indicated that 25 of these genes functioned to promote efficient HR repair, thus representing a comprehensive HR gene set in fission yeast. Included in this gene set are 10 genes not previously implicated in HR repair; nse5⁺, nse6⁺, ddb1⁺, cdt2⁺, alm1⁺, snz1⁺, kin1⁺, pal1⁺, SPAC31G5.18c⁺ and SPCC613.03⁺. Detailed characterisation of ddb1Δ and cdt2Δ established a role for the Ddb1-Cul4Cdt2 ubiquitin ligase complex in HR. The findings presented here support a model in which break-induced Rad3 and Ddb1-Cul4Cdt2 ubiquitin ligase-dependent Spd1 degradation promotes ribonucleotide reductase activation and nucleotide biosynthesis, which is required for post-synaptic ssDNA gap filling during HR repair. Lastly, the role of HR genes in suppressing chromosome loss and rearrangements was examined. A striking inverse correlation between levels of gene conversion and levels of both chromosome loss and LOH was observed across the HR gene deletion set. These findings support a common and likely evolutionarily conserved role for HR genes in suppressing both chromosome loss and break-induced chromosomal rearrangements resulting from extensive end processing associated with failed HR repair.