Functions of the haspin genes in the control of budding yeast cell cycle Haspin is a serine/threonine kinase, originally identified in mouse and human cells. Haspin-like proteins have been found in all eukaryotic organisms, including yeast the Saccharomyces cerevisiae. In budding yeast there are two haspin-like homologues encoded by the genes ALK1 and ALK2, these two ORFs are previously uncharacterized. We identified Alk1 as an interactor of the DNA damage checkpoint protein Ddc1 in a two hybrid screening, also Alk2 interacts with Ddc1 in the same system. The first part of my thesis work consisted in the biochemical characterization of the Alk1 and Alk2 proteins and their cell cycle regulation. To gain some insight in the in vivo function of Alk1 and Alk2, we decided to use two classic genetic experimental approach: overexpression and deletion of ALK1 and ALK2 genes. While ALK1 overexpression does not affect cell cycle progression, the overexpression of ALK2 leads to accumulation of cells with a 2C DNA content and undivided nuclei. We found that cohesin cleavage induced by the TEV protease, bypasses these phenotypes. It is possible that Alk2 plays a direct action on the cohesin complex or, on the separase. Our results suggest, a role for Alk2 in the establishment of chromosomal cohesion, in the control of cohesin stability and in the regulation of separase. The second approach was based on a classical genetic analysis of deletion mutants. Dalk1 and Dalk2 strains did not show any sensitivity of single and double mutants to genotoxic agents, or defects in DNA damage checkpoint activation. Later, by analysing the phenotype of ALK2 overexpression, a mitotic role for Alk2 was emerged, and we thus decided to focus our attention on the role of Alk1 and Alk2 in mitosis. On plates Dalk1Dalk2 cells show sensitivity to benomyl treatment, this result orients our study on the role of haspins toward the spindle dynamics field. We first test the role of Alk1 and Alk2 in the regulation of the spindle checkpoint but our results seem to exclude a role for Alk1 and Alk2. We tested whether Dalk1Dalk2 cells are capable to re-assemble and re-orient the mitotic spindle correctly after microtubule depolymerization, in Dalk1Dalk2 cells this process seems to takes place with normal timing.
|Titolo:||Functions of the haspin genes in the control of budding yeast cell cycle|
|Supervisori e coordinatori interni:||MANTOVANI, ROBERTO|
|Data di pubblicazione:||2008|
|Settore Scientifico Disciplinare:||Settore BIO/11 - Biologia Molecolare|
|Citazione:||Functions of the haspin genes in the control of budding yeast cell cycle ; R. Mantovani, P. Plevani. - Milano : Università degli studi di Milano. DIPARTIMENTO DI SCIENZE BIOMOLECOLARI E BIOTECNOLOGIE, 2008. ((20. ciclo, Anno Accademico 2006/2007.|
|Appare nelle tipologie:||13 - Tesi di dottorato discussa entro ottobre 2010|