Metastasis is the spread of cancer cells from the primary tumor to distant sites. It is the most dangerous attribute of cancer, and also the principle cause of cancerrelated morbidity and mortality. Metastasis suppressor genes are a group of genes that suppress tumor metastasis without significant effect on tumorigenicity. NM23 was the first identified metastasis suppressor gene, and loss of its expression is a frequent hallmark of metastatic growth in multiple cancers (e.g. melanoma, carcinomas of breast, stomach and liver). NM23-H1 possesses at least three enzymatic activities, including nucleoside diphosphate kinase (NDPK), histidine kinase (hisK), and a more recently described 3f-5f exonuclease (EXO). While the hisK has been shown to be linked to the suppression of cell motility, the NDPK has been reported to be unrelated to the suppression of metastatic potential indirectly. Relevance of EXO has not been addressed. Other known 3f-5f exonuclease are closely associated with DNA repair functions, suggesting NM23-H1 may suppress mutations required for metastasis. As a transcription factor, NM23 has been shown to modestly downregulate the transcription on PDGF-A chain, a growth factor oncogene, either alone or in association with another transcriptional factor, Pur@. At the same time, identification of NM23-H1 as a 3f-5fexonuclease suggests the role of NM23-H1 in DNA repair. Etoposide and cisplatin elicited nuclear translocation of H1 within 4 h in HeLa and HepG2 cells, seen as accumulation of H1 in small intranuclear foci, strongly suggesting the DNA repair function of H1. To investigate the enzymatic function contributing to metastasis suppressor activity of H1, complementation system was used by transfecting NM23-H1 with individually disrupted enzymatic function into 2 melanoma cell lines, 1205LU and WM793. Overexpression of H1 in 1205LU suppressed lung metastasis in vivo without effect on indices of transformation (e.g. proliferation, soft agar colonization). EXO- deficient H1 and NDPK-deficient H1 lost suppression of lung metastasis, while hisK-deficient H1 maintained suppressor activity. Consistent with the results in 1205LU cells, EXO-deficient H1 and NDPKdeficient H1 lost suppression of the progression of WM793 cells in protein-free medium, while WT and hisK-deficient H1 prevented the progression. Taken together, these data suggest that the NDPK and/or 3f-5fEXO activity of H1 inhibits the progression of premetastatic cells to the metastatic phenotype, possibly via a DNA repair function or other structural transactions with DNA.

Maintaining genomic integrity is important for any organism. DNA mismatch repair (MMR) serves to correct errors that occur during DNA replication and recombination, such as unpaired bases or mismatched bases. Mutl is a key player and serves to coordinate protein-protein interactions. Recently it has been shown that human Mutl functions as an endonuclease and that this activity is imperative for functioning MMR. In this work, the X-ray crystal structure of the C-terminal endonuclease domain of Bacillus subtilis Mutl (BsMutL-CTD) is presented. Diffraction quality crystals of BsMutL-CTD were grown using vapor diffusion. The crystal structure of BsMutL-CTD was solved using multiwavelength anomalous diffraction. The structure reveals a putative metal binding site which clusters closely in space with endonuclease motif. Using the structure and sequence homology, several mutations were made and an investigation into the endonuclease activity of BsMutL was performed. BsMutL was confirmed to be a manganese-dependent endonuclease and key residues which contribute to endonuclease function were identified. / Thesis / Master of Science (MSc)

Adult Stem Cells (SCs) have been found in almost every organ. They are responsible for<p>homeostasis and tissue repair after injury. SCs reside and self-renew in the adult body<p>throughout the life of the organism. In rapid self-renewing organs, such as the skin, the<p>intestine and the blood, SCs divide many times during the life of the animal in order to sustain<p>the homeostatic needs of the tissue.<p>All cells of the body, including SCs, are constantly subjected to DNA assaults arising from<p>endogenous sources, such as reactive oxygen species (ROS) generated by cellular<p>metabolism, or exogenous assaults arising from the environment. The DNA damage response<p>(DDR) and DNA repair mechanisms protect cells from accumulating DNA damage by<p>inducing transient cell cycle arrest allowing DNA repair, triggering senescence or apoptosis.<p>DNA damages trigger the activation of the effectors of the DDR inducing a transient cell<p>cycle arrest, allowing DNA repair, or triggering a permanent arrest of the cell cycle or<p>apoptosis if damages are too extensive.<p>As skin is the outermost barrier of the body, epidermal cells, including SCs, are<p>continuously subjected to genotoxic stress, such as UV rays, ionizing radiation (IR) and<p>chemicals. The skin epidermis is composed of hair follicles (HFs), its associated sebaceous<p>gland (SG) and the surrounding inter-follicular epidermis (IFE). Different types of SCs<p>maintain the homeostasis of the skin; multipotent adult bulge SCs ensure the cyclic<p>regeneration of the HF and the repair of the epidermis after injury, while individual unipotent<p>SCs ensure homeostasis of the SG and the IFE.<p>In tissues with high cellular turnover, such as the epidermis, the numerous divisions that a<p>SC undergoes could result in the accumulation of replication-associated DNA damage. It has<p>been suggested that adult SCs may undergo asymmetric divisions in which the daughter SC<p>retains the older (thus “immortal”) DNA strand, while the daughter cell committed to<p>differentiation inherits the newly synthesized strand that may have incorporated replicationderived<p>mutations. The in vivo relevance of this mechanism is still a matter of intense debate.<p>We used multiple in vivo experimental approaches to investigate precisely how bulge SCssegregate their chromosomes during HF morphogenesis, SC activation and skin homeostasis.<p>Using pulse-chase experiments with two different uridine analogs together with DNAindependent<p>chromatin labelling, we showed that multipotent HF SCs segregate their<p>chromosomes randomly, and that the label-retention observed in the skin epidermis derives<p>solely from relative quiescence of skin SCs 1.<p>We investigated the in vivo response of multipotent adult HF bulge SCs to DNA damage<p>induced by IR. We showed that bulge SCs are profoundly resistant to DNA damage-induced<p>cell death compared to their more mature counterparts. Interestingly, we demonstrated that<p>resistance of bulge SCs to IR-induced apoptosis does not rely on their relative quiescence.<p>Moreover, we showed that DDR in SCs does not lead to premature senescence. We found that<p>two intrinsic cellular mechanisms participate in the resistance of bulge SCs to DNA damageinduced<p>cell death. Bulge SCs express higher level of the anti-apoptotic Bcl-2 and present<p>more transient activation of p53 due to a faster DNA repair activity mediated by a nonhomologous<p>end joining (NHEJ) mechanism. Since NHEJ is not error free, this property<p>might be a double-edged sword, supporting short-term survival of bulge SCs but impairing<p>long-term genomic integrity 2.<p>While we unveiled the relevance of DSBs repair by NHEJ in the skin epidermis, little is<p>known about the role of homologous recombination (HR) during the morphogenesis of the<p>skin epidermis. Brca1 is an essential protein for HR. Conditional deletion of Brca1 in the<p>developing epidermis leads to congenital alopecia accompanied by a decreased density of hair<p>placodes. The remaining HFs never produce mature hair and progressively degenerate due to<p>high levels of apoptosis. Multipotent adult HF bulge SCs cannot be detected in adult HF in<p>the Brca1 cKO epidermis. Brca1 deletion in the epidermis triggers p53 activation throughout<p>the epidermis, which activates apoptosis. Interestingly, IFE and the isthmus region of the HF<p>do not present any pathological phenotype by constitutive deletion of Brca1. Our results<p>demonstrated the critical role of Brca1 during HF morphogenesis. Future studies will be<p>required to understand the molecular mechanisms controlling this phenotype / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

O mecanismo pelo qual uma célula responde a algum dano no seu material genético é extremamente importante. Isto ocorre pela rápida ativação da maquinaria de reparo de danos no DNA, a qual é composta por uma rede intrincada de sinalização proteica, culminando no reparo do DNA; porém se o dano for irreparável ocorre ativação de mecanismos de morte celular. RhoA,e Rac1 pertencem a família das pequenas proteínas sinalizadoras Rho GTPases, as quais atuam como interruptores moleculares ciclando entre estado ativo (ligada a GTP) e inativo (ligada a GDP). Os componentes desta família estão relacionados ao controle dos mais diversos processos celulares como, por exemplo, remodelamento do citoesqueleto, migração, adesão, endocitose, progressão do ciclo celular e oncogênese. No entanto, apesar das proteínas Rho GTPases estarem envolvidas em um amplo espectro de atividades biológicas, há poucas informações sobre seu papel na manutenção da integridade genômica quando células são submetidas a algum agente genotóxico. Para investigar o envolvimento das GTPases RhoA e Rac1 nas respostas de células submetidas a radiação gama, foram gerados, a partir de células de carcinoma de cervix humano - HeLa, sublinhagens clonais mutantes de RhoA e Rac1 expressando exogenamente RhoA constitutivamente ativa (HeLa-RhoA V14), RhoA dominante negativa (HeLa-RhoA N19), Rac1 constitutivamente ativa (HeLa-Rac1 V12) e Rac1 dominante negativa (HeLa-Rac N17). Após estas linhagens celulares serem expostas a diferentes doses de radiação gama, observamos que ambas GTPases, RhoA e Rac1, são ativadas em resposta aos efeitos da radiação. Além disso, a modulação da atividade destas enzimas, através das mutações, levou a uma alteração das respostas celulares frente aos danos no DNA, como uma redução da capacidade de reparar quebras simples e duplas nas fitas do DNA. Por outro lado, a deficiência de RhoA ou Rac1 GTPase levou a uma redução da ativação de Chk1 e Chk2 ou da fosforilação da histona H2AX, respectivamente, prejudicando os mecanismos de detecção de danos no DNA e levando as células a permanecerem mais tempo nos pontos de checagem G1/S e/ou G2/M do ciclo celular. Esses fatores contribuíram de modo expressivo para a redução da proliferação e sobrevivência celular levando as células à morte. Por fim, ensaios celulares de reparo de danos de um DNA exógeno através de mecanismos de Recombinação Homóloga (HR) e Recombinação Não-Homóloga de extremidades (NHEJ), demonstraram que a inibição da atividade de RhoA reduz significativamente a eficiência de ambas vias de reparo. Desta maneira, este trabalho demonstra e reforça a existência de mais um viés de atuação das pequenas GTPases RhoA e Rac1, agora em células HeLa, nas respostas celulares aos danos induzidos por exposição a radiação gama, modulando a sobrevivência, proliferação e indiretamente modulando resposta ao reparo do DNA através da via de Recombinação Homóloga e Não-Homóloga / The mechanism by which a cell responds to DNA damage is extremely important. This occurs by a quick activation of the DNA damage repair machinery, which consists of an intricate protein signaling network culminating in DNA repair. But if the damages are irreparable occurs there is activation of cell death mechanisms. RhoA and Rac1 belong to family of small Rho GTPases, signaling proteins that act as molecular switches cycling between the active state (GTP-bound) and inactive state (GDP-bound). Members of this family are implicated in the control of diverse cellular process such as cytoskeletal remodeling, migration, adhesion, endocytosis, cell cycle progression, and oncogenesis. However, despite Rho proteins are involved in a broad spectrum of biological activities, there is just a few information about their roles in the maintenance of genomic integrity, that is, when the cells are subjected to some kinf of genotoxic agent. To investigate the involvement of the GTPases RhoA and Rac1 in cellular responses to gamma radiation, we generated from human cervix carcinoma cells - HeLa, clonal sublines of RhoA and Rac1 mutants, exogenous and stably expressing the constitutively active RhoA (HeLa-RhoA V14), the dominant negative RhoA (HeLa-RhoA N19), the constitutively active Rac1 (HeLa-Rac1 V12) and the dominant negative Rac1 (HeLa-Rac1 N17). After all these cell lines have been exposed to different doses of gamma radiation, we found that both GTPases, RhoA and Rac1, are activated in response to the radiation effects. Furthermore, the modulation of two enzymes activity, by using the mutant clones, led to a change in cellular responses to the DNA damage, as the reduction in the capacity of repairing DNA single and double strand breaksr. On the other hand, the deficiency of RhoA or Rac1 GTPase led to a reduction of Chk1 and Chk2 activation, or on the phosphorylation of histone H2AX, respectively, hindering the mechanisms of DNA damage detection and arresting cells in the G1/S and/or G2/M checkpoints of cell cycle. These factors significantly contributed to the reduction of cell proliferation and survival, leading cells to death. Finally, cellular assays of DNA damage repair of exogenous DNA by Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ), demonstrated that RhoA inhibition significantly reduced the repair efficiency of both pathways. Thus, this work demonstrates and reinforces the existence of other biological functions of small GTPases RhoA and Rac1 in HeLa cells, by regulating cellular responses to DNA damage induced by exposure to gamma radiation, modulating the survival, proliferation and indirectly modulating the response to DNA damage repair pathway through the Homologous Recombination and Non-Homologous Recombination

O mecanismo pelo qual uma célula responde a algum dano no seu material genético é extremamente importante. Isto ocorre pela rápida ativação da maquinaria de reparo de danos no DNA, a qual é composta por uma rede intrincada de sinalização proteica, culminando no reparo do DNA; porém se o dano for irreparável ocorre ativação de mecanismos de morte celular. RhoA,e Rac1 pertencem a família das pequenas proteínas sinalizadoras Rho GTPases, as quais atuam como interruptores moleculares ciclando entre estado ativo (ligada a GTP) e inativo (ligada a GDP). Os componentes desta família estão relacionados ao controle dos mais diversos processos celulares como, por exemplo, remodelamento do citoesqueleto, migração, adesão, endocitose, progressão do ciclo celular e oncogênese. No entanto, apesar das proteínas Rho GTPases estarem envolvidas em um amplo espectro de atividades biológicas, há poucas informações sobre seu papel na manutenção da integridade genômica quando células são submetidas a algum agente genotóxico. Para investigar o envolvimento das GTPases RhoA e Rac1 nas respostas de células submetidas a radiação gama, foram gerados, a partir de células de carcinoma de cervix humano - HeLa, sublinhagens clonais mutantes de RhoA e Rac1 expressando exogenamente RhoA constitutivamente ativa (HeLa-RhoA V14), RhoA dominante negativa (HeLa-RhoA N19), Rac1 constitutivamente ativa (HeLa-Rac1 V12) e Rac1 dominante negativa (HeLa-Rac N17). Após estas linhagens celulares serem expostas a diferentes doses de radiação gama, observamos que ambas GTPases, RhoA e Rac1, são ativadas em resposta aos efeitos da radiação. Além disso, a modulação da atividade destas enzimas, através das mutações, levou a uma alteração das respostas celulares frente aos danos no DNA, como uma redução da capacidade de reparar quebras simples e duplas nas fitas do DNA. Por outro lado, a deficiência de RhoA ou Rac1 GTPase levou a uma redução da ativação de Chk1 e Chk2 ou da fosforilação da histona H2AX, respectivamente, prejudicando os mecanismos de detecção de danos no DNA e levando as células a permanecerem mais tempo nos pontos de checagem G1/S e/ou G2/M do ciclo celular. Esses fatores contribuíram de modo expressivo para a redução da proliferação e sobrevivência celular levando as células à morte. Por fim, ensaios celulares de reparo de danos de um DNA exógeno através de mecanismos de Recombinação Homóloga (HR) e Recombinação Não-Homóloga de extremidades (NHEJ), demonstraram que a inibição da atividade de RhoA reduz significativamente a eficiência de ambas vias de reparo. Desta maneira, este trabalho demonstra e reforça a existência de mais um viés de atuação das pequenas GTPases RhoA e Rac1, agora em células HeLa, nas respostas celulares aos danos induzidos por exposição a radiação gama, modulando a sobrevivência, proliferação e indiretamente modulando resposta ao reparo do DNA através da via de Recombinação Homóloga e Não-Homóloga / The mechanism by which a cell responds to DNA damage is extremely important. This occurs by a quick activation of the DNA damage repair machinery, which consists of an intricate protein signaling network culminating in DNA repair. But if the damages are irreparable occurs there is activation of cell death mechanisms. RhoA and Rac1 belong to family of small Rho GTPases, signaling proteins that act as molecular switches cycling between the active state (GTP-bound) and inactive state (GDP-bound). Members of this family are implicated in the control of diverse cellular process such as cytoskeletal remodeling, migration, adhesion, endocytosis, cell cycle progression, and oncogenesis. However, despite Rho proteins are involved in a broad spectrum of biological activities, there is just a few information about their roles in the maintenance of genomic integrity, that is, when the cells are subjected to some kinf of genotoxic agent. To investigate the involvement of the GTPases RhoA and Rac1 in cellular responses to gamma radiation, we generated from human cervix carcinoma cells - HeLa, clonal sublines of RhoA and Rac1 mutants, exogenous and stably expressing the constitutively active RhoA (HeLa-RhoA V14), the dominant negative RhoA (HeLa-RhoA N19), the constitutively active Rac1 (HeLa-Rac1 V12) and the dominant negative Rac1 (HeLa-Rac1 N17). After all these cell lines have been exposed to different doses of gamma radiation, we found that both GTPases, RhoA and Rac1, are activated in response to the radiation effects. Furthermore, the modulation of two enzymes activity, by using the mutant clones, led to a change in cellular responses to the DNA damage, as the reduction in the capacity of repairing DNA single and double strand breaksr. On the other hand, the deficiency of RhoA or Rac1 GTPase led to a reduction of Chk1 and Chk2 activation, or on the phosphorylation of histone H2AX, respectively, hindering the mechanisms of DNA damage detection and arresting cells in the G1/S and/or G2/M checkpoints of cell cycle. These factors significantly contributed to the reduction of cell proliferation and survival, leading cells to death. Finally, cellular assays of DNA damage repair of exogenous DNA by Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ), demonstrated that RhoA inhibition significantly reduced the repair efficiency of both pathways. Thus, this work demonstrates and reinforces the existence of other biological functions of small GTPases RhoA and Rac1 in HeLa cells, by regulating cellular responses to DNA damage induced by exposure to gamma radiation, modulating the survival, proliferation and indirectly modulating the response to DNA damage repair pathway through the Homologous Recombination and Non-Homologous Recombination

Maintenance of genomic integrity with high fidelity is of prime importance to any organism. An insult which may result in compromised genome integrity is prevented or its consequences are monitored by advanced cellular networks, collectively called the DNA damage response (DDR). Various DNA repair pathways, which are part of DDR, constantly correct the genome in the event of any undesirable change in the genetic material and prevent the transmission of any impairment to daughter cells. Non homologous DNA end joining (NHEJ) is the predominant DNA repair pathway associated with DDR in higher eukaryotes, correcting double-strand breaks (DSBs). Microhomology mediated end joining (MMEJ), an alternate mechanism to NHEJ also exists in cells, which is associated with erroneous joining of broken DNA, leading to mutagenesis. DDR is of paramount importance in cellular viability and therefore, any defects in DDR or the imbalance of repair pathways contribute to mutations, cellular transformations and various neurodegenerative and congenital abnormalities. Here, we investigate the DDR via NHEJ and MMEJ pathways during embryonic development in mice as well as in presence of an environmental pollutant, Endosulfan, in order to understand how physiological and exogenous factors condition the balance of repair pathways. Among various classes of pesticides known to cause side effects, organochlorine pesticides (OCPs) lead the list, possessing high transport potential, and a variety of toxic and untoward health effects. Endosulfan is a widely used organochlorine pesticide and is speculated to be detrimental to human health. However, very little is known about mechanism of its genotoxicity. Using in vivo and ex vivo model systems, we showed that exposure to Endosulfan induced reactive oxygen species (ROS) in a concentration dependent manner. Using an array of assays and equivalents of sub-lethal concentrations comparable to the detected level of Endosulfan in humans living in active areas of exposure, we demonstrated that ROS production by Endosulfan resulted in DNA double-strand breaks in mice, rats and human cells. In mice, the DNA damage was predominantly detected in type II pneumocytes of lung tissue; spermatogonial mother cells and primary spermatids of testes. Importantly, Endosulfan-induced DNA damage evoked DDR, which further resulted in elevated levels of classical NHEJ. However, sequence analyses of NHEJ junctions revealed that Endosulfan treatment resulted in extensive processing of broken DNA, culminating in increased and long junctional deletions, thereby favouring erroneous repair. We also find that exposure to Endosulfan led to significantly increased levels of MMEJ, which is a LIGASE III dependent, alternative, non classical repair pathway, encompassing long deletions and processing of DNA. Further, we show that the differential expression of proteins following exposure to Endosulfan correlated with activation of alternative DNA repair. At the physiological level, using mouse model system, we showed that exposure to Endosulfan affected physiology and cellular architecture of organs and tissues. Among all organs, damage to testes was extensive and it resulted in death of different testicular cell populations. We also found that the damage in testes resulted in qualitative and quantitative defects during spermatogenesis in a time dependent manner, increasing epididymal ROS levels and affecting sperm chromatin integrity. This further culminated in reduced number of epididymal sperms and actively motile sperms, which finally resulted in reduced fertility in male but not in female mice. Repair of DSBs is important for maintaining genomic integrity during the successful development of a fertilized egg into a whole organism. To date, the mechanism of DSB repair in post implantation embryos has been largely unknown except for the differential requirement of DNA repair genes in the course of development. These studies relied on null mutation analysis of animal phenotypes and therefore a quantitative understanding of repair pathways was absent. In the present study, using a cell free repair system derived from different embryonic stages of mice, we found that canonical NHEJ is predominant at 14.5 day of embryonic development. Interestingly, all types of DSBs tested were repaired by LIGASE IV/XRCC4 and Ku-dependent classical NHEJ. Characterization of end-joined junctions and expression studies further showed evidence for C-NHEJ. Strikingly, we observed non canonical end joining accompanied by DSB resection, dependent on microhomology and LIGASE III in 18.5-day embryos. Further we observed an elevated expression of CtIP, MRE11, and NBS1 at this stage, suggesting that it could act as a switch between classical and microhomology-mediated end joining at later stages of embryonic development. Keeping these observations in mind, we wondered if Endosulfan affected the differential regulation of DDR during development, similar to mice tissues. Upon analysing the effect of endosulfan on NHEJ/MMEJ at above mentioned stages of mouse embryonic development, we found that C-NHEJ efficiency remained low or unaltered while the efficiency of MMEJ was upregulated significantly, perturbing the repair balance during embryo development and hence facilitating mutagenic repair. Thus, our results establish the existence of both classical and non classical NHEJ pathways during the post implantation stages of mammalian embryonic development. Our studies also provide deeper insights into physiological and molecular events leading to male infertility upon Endosulfan exposure and its impact on impairing the differential regulation of DNA repair during embryonic development. Our findings suggest the plasticity of DNA repair pathways in physiological and pathological conditions and provide insights into mechanism of genome instability due to DNA repair imbalance, when exposed to environmental mutagens.