title: Regulation of tolerance to DNA alkylating damage by Dot1 and Rad53 in Saccharomyces cerevisiae.
abstract: To maintain genomic integrity cells have to respond properly to a variety of exogenous and endogenous factors that produce genome injuries and interfere with DNA replication. DNA integrity checkpoints coordinate this response by slowing cell cycle progression to provide time for the cell to repair the damage, stabilizing replication forks and stimulating DNA repair to restore the original DNA sequence and structure. In addition, there are also mechanisms of damage tolerance, such as translesion synthesis (TLS), which are important for survival after DNA damage. TLS allows replication to continue without removing the damage, but results in a higher frequency of mutagenesis. Here, we investigate the functional contribution of the Dot1 histone methyltransferase and the Rad53 checkpoint kinase to TLS regulation in Saccharomyces cerevisiae. We demonstrate that the Dot1-dependent status of H3K79 methylation modulates the resistance to the alkylating agent MMS, which depends on PCNA ubiquitylation at lysine 164. Strikingkly, either the absence of DOT1, which prevents full activation of Rad53, or the expression of an HA-tagged version of RAD53, which produces low amounts of the kinase, confer increased MMS resistance. However, the dot1Δ rad53-HA double mutant is hypersensitive to MMS and shows barely detectable amounts of activated kinase. Furthermore, moderate overexpression of RAD53 partially suppresses the MMS resistance of dot1Δ. In addition, we show that MMS-treated dot1Δ and rad53-HA cells display increased number of chromosome-associated Rev1 foci. We propose that threshold levels of Rad53 activity exquisitely modulate the tolerance to alkylating damage at least by controlling the abundance of the key TLS factor Rev1 bound to chromatin.
authors.affiliation: Instituto de Microbiología Bioquímica, Consejo Superior de Investigaciones Científicas and University of Salamanca, 37007 Salamanca, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: Conde
authors.forename: David
authors.initials: D
authors.lastname: Ontoso
authors.forename: Isabel
authors.initials: I
authors.lastname: Acosta
authors.forename: Alfonso
authors.initials: A
authors.lastname: Gallego-Sánchez
authors.forename: Avelino
authors.initials: A
authors.lastname: Bueno
authors.forename: Pedro A
authors.initials: PA
authors.lastname: San-Segundo
mesh_terms: D000477:Alkylating Agents / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics* / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D008741:Methyl Methanesulfonate / Q000378:metabolism; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000477:Alkylating Agents; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D006657:Histones; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D008741:Methyl Methanesulfonate; D000076983:Histone Methyltransferases; C416235:Dot1 protein, S cerevisiae; D011495:Histone-Lysine N-Methyltransferase; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae