DNA replication is a highly complex part of cell metabolism that ensures safe propagation of the genome through tight regulation of the expression, localization, and activity of a large number of factors. Replication starts from distinct sites in the genome and initiation events are temporally ordered in a manner that is, on average, highly reproducible across cell populations. The specific order with which different parts of the genome are replicated has been proposed to be important to processes such as gene expression, cell differentiation, development, and genome evolution. Nevertheless, the fundamental mechanisms that are responsible for establishing these timing programs remain elusive. Unlike in higher eukaryotes, DNA replication in budding yeast initiates at sequence-specific loci called origins of replication. The timing of initiation at these loci is determined by the activation of the main replicative helicase Minichromosome Maintenance (MCM) complex. Recent results have placed MCM in a key role in establishing a replication timing program that is reproducible but arises from stochastic activation of origins, as has been observed in yeast and higher eukaryotes. One particular model posits that the loading of multiple MCMs at individual origins increases the chances that origins will be activated earlier in S phase by a limited amount of initiation factors. To further test this model, we set out to examine the consequences of modulating MCM levels in budding yeast in order to ascertain their effects on the dynamics of helicase loading during G1 and subsequent replication timing. Overexpression of MCM2-7 had no effects on cell viability, cell cycle progression, MCM abundance at origins, or replication timing. On the other hand, depletion of Mcm4, one of the six obligate components of the MCM helicase, caused reduced viability, slower progression through S phase, and increased sensitivity to replication stress. Importantly, Mcm4 depletion led to differential reduction in MCM loading at origins during G1, with low MCM origins being disproportionately affected by reduced MCM pools. Finally, reduced MCM loading at origins of replication led to delayed replication during S phase. Our data support a model where the loading activity of origins, controlled by their ability to recruit ORC and compete for MCMs, determines the number of helicases loaded, which in turn has strong implications for replication timing.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-05-12T13:22:54Z No. of bitstreams: 1 Manuela Torres Andion Vidal Estudo...2015.pdf: 4528048 bytes, checksum: dbdb598bed619445f96ad9d571f8324e (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-05-12T13:23:08Z (GMT) No. of bitstreams: 1 Manuela Torres Andion Vidal Estudo...2015.pdf: 4528048 bytes, checksum: dbdb598bed619445f96ad9d571f8324e (MD5) / Made available in DSpace on 2015-05-12T13:23:08Z (GMT). No. of bitstreams: 1 Manuela Torres Andion Vidal Estudo...2015.pdf: 4528048 bytes, checksum: dbdb598bed619445f96ad9d571f8324e (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / O adenoma pleomórfico (AP), o carcinoma mucoepidermóide (CME) e o carcinoma adenóide cístico (CAC) representam tumores frequentes em glândula salivar. A via de sinalização Sonic Hedgehog (Hh) e o Transdutor de sinal e ativador da transcrição 3 (STAT3) desempenham funções importantes na proliferação celular, favorecendo o desenvolvimento tumoral e a proteína MCM3 tem sido considerada uma nova classe de marcadores de proliferação celular. Portanto, o presente trabalho propõe-se a estudar componentes da via Hh, bem como o STAT3 e o MCM3 em neoplasias de glândula salivar, na tentativa de adicionar informações sobre as características biológicas dessas neoplasias. Foram utilizados 9 casos de AP, 17 casos de CAC e 20 casos de CME e, por meio da técnica imunoistoquímica, realizou-se a detecção das seguintes proteínas: SHH, GLI1, SUFU, HHIP, STAT3 e MCM3. No AP, observou-se alta expressão citoplasmática de SHH e SUFU, e baixa expressão de STAT3 e MCM3. No CAC, observou-se alta expressão de GLI1, HHIP e STAT3 e baixa expressão de SHH, SUFU e MCM3. No CME, observou-se alta expressão de SHH, GLI1, SUFU e HHIP e baixa expressão de STAT3 e MCM3. Quando comparado entre os tipos tumorais, observou-se diferença estatisticamente significante para expressão de SHH (p=0.0064), STAT3 (p=0.0003) e MCM3 (p=0.0257). Ao comparar a marcação parenquimal e estromal, observou-se maior expressão em parênquima para todos os tumores (p<0.05). Em glândula salivar normal, observou-se marcação citoplasmática das células ductais para SHH, GLI1, SUFU e HHIP, uma discreta marcação citoplasmática para STAT3 e ausência de marcação para MCM3. Não foi observada correlação entre a via Hh e STAT3 nos tumores de glândula salivar (p>0.05). Os resultados desse trabalho apontam para uma possível participação da via Hh na morfogênese e no desenvolvimento do AP, CAC e CME, assim como a participação do STAT3 no desenvolvimento do CAC. Em adição, o MCM3 não parece ser um bom marcador de proliferação para tumores de glândula salivar. Sugerimos que novos estudos sejam realizados visando compreender o mecanismo pelo qual a via Hh e o STAT3 promovem o crescimento e progressão desses tumores e identificar inibidores dessas vias. / The pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC) and the adenoid cystic carcinoma (ACC) are common tumors arising from salivary glands. The Sonic Hedgehog signaling pathway (Hh) and signal transducer and activator of transcription 3 (STAT3) play important roles in cell proliferation, favoring tumor growth. The MCM3 protein has been considered as a novel class of cell proliferation markers. The aim of this investigation was to study components of the Hh pathway, as well as STAT3 and MCM3 in salivary gland neoplasms in an attempt to add information about the biological characteristics of these neoplasms. We used 9 cases of PA, 17 cases of ACC and 20 cases of MEC. Using immunohistochemistry, were investigated: SHH, GLI1, Sufu, HHIP, STAT3 and MCM3. In PA, there was high expression of cytoplasmic SHH and Sufu, and low expression of STAT3 and MCM3. In the ACC, there was high expression of GLI1, HHIP and STAT3 and low expression of SHH, SUFU and MCM3. In the MEC, we observed high expression of SHH, GLI1, SUFU and HHIP and low expression of STAT3 and MCM3. There was a statistically significant difference between SHH (p=0.0064), STAT3 (p=0.0003) and MCM3 (p=0.0257) when all tumors were compared. And a higher expression in parenchyma for all tumors when stroma and parenchyma were compared (p<0.05). In normal salivary gland, ductal segment showed imunolabeling for SHH, GLI1, SUFU and HHIP, a discrete cytoplasmic labeling for STAT3 and MCM3 was negative. No correlation was observed between the Hh pathway and STAT3 in salivary gland tumors (p>0.05). The findings suggest a possible role of Hh pathway in the morphogenesis and development of AP, CAC and CME, as well as the participation of STAT3 in the development of ACC. In addition, the MCM3 did not seem to be a good marker of proliferation for salivary gland tumors. It is important that further studies be conducted to understand the mechanism by which the Hh pathway and the STAT3 promote the growth and progression of these tumors and inhibitors of these pathways might be identified.