THE ROLE OF THE S. CEREVISIAE SUMO PROTEASE ULP2 IN DNA REPLICATION AND GENOME INTEGRITY

Sumoylation has emerged as an important player in several biological processes involved in the maintenance of genome stability. A number of proteins implicated in DNA metabolism-associated processes, such as DNA replication, repair and chromosome segregation, were shown to sumoylated. Moreover, alterations in protein sumoylation and desumoylation, caused by deregulation of enzymes involved in the SUMO pathway, were associated with cancer development. Work done previously in our laboratory and by other groups suggested an important role for sumoylation in promoting DNA damage tolerance. However, the molecular mechanisms by which DNA damage regulates sumoylation and how balance between sumoylation and desumoylation impinges the cellular response to genotoxic stress are questions that need to be clarified. Different scenarios could account for regulating sumoylation, one of which envisages a control over the activity of the desumoylating enzymes. Budding yeast has two desumoylating enzymes, Ulp1 and Ulp2, which have distinct cellular localization and enzymatic activities. Starting by investigating whether Ulp1 and/or Ulp2 undergo any type of functional regulation, I found the nuclear SUMO protease Ulp2 to be phosphorylated both in a cell cycle-dependent manner and in response to replication stress or DNA damage. The present work aimed at studying the molecular mechanisms through which Ulp2 promotes genome integrity, both in physiological conditions and in conditions of replication stress. By examining the genome-wide localization of Ulp2 by ChIP-on-chip I observed that Ulp2 is bound to centromeric regions throughout the cell cycle and this enrichment is extended to pericentromeric regions in cells arrested at metaphase. Lack of Ulp2 affects the chromatin structure at centromeres and nearby regions. Following replication stress, Ulp2 is bound to most of the origins of replication that are active under our experimental conditions and is required for efficient origin firing. Also under conditions of replication stress Ulp2 is important for the maintenance of chromatin structure and for the binding of histone variants that are known to play a role in damage tolerance to origins of replication. Thus, Ulp2 appears to play key roles in regulating the status of chromatin to make it suitable for efficient replication and chromosome segregation.