title: Sak1 kinase interacts with Pso2 nuclease in response to DNA damage induced by interstrand crosslink-inducing agents in Saccharomyces cerevisiae.
abstract: By isolating putative binding partners through the two-hybrid system (THS) we further extended the characterization of the specific interstrand cross-link (ICL) repair gene PSO2 of Saccharomyces cerevisiae. Nine fusion protein products were isolated for Pso2p using THS, among them the Sak1 kinase, which interacted with the C-terminal β-CASP domain of Pso2p. Comparison of mutagen-sensitivity phenotypes of pso2Δ, sak1Δ and pso2Δsak1Δ disruptants revealed that SAK1 is necessary for complete WT-like repair. The epistatic interaction of both mutant alleles suggests that Sak1p and Pso2p act in the same pathway of controlling sensitivity to DNA-damaging agents. We also observed that Pso2p is phosphorylated by Sak1 kinase in vitro and co-immunoprecipitates with Sak1p after 8-MOP+UVA treatment. Survival data after treatment of pso2Δ, yku70Δ and yku70Δpso2Δ with nitrogen mustard, PSO2 and SAK1 with YKU70 or DNL4 single-, double- and triple mutants with 8-MOP+UVA indicated that ICL repair is independent of YKu70p and DNL4p in S. cerevisiae. Furthermore, a non-epistatic interaction was observed between MRE11, PSO2 and SAK1 genes after ICL induction, indicating that their encoded proteins act on the same substrate, but in distinct repair pathways. In contrast, an epistatic interaction was observed for PSO2 and RAD52, PSO2 and RAD50, PSO2 and XRS2 genes in 8-MOP+UVA treated exponentially growing cells.
authors.affiliation: Biotechnology Center, Federal University of Rio Grande do Sul (UFRGS), 91507-970 Porto Alegre, RS, Brazil.
authors.forename: Fernanda M
authors.initials: FM
authors.lastname: Munari
authors.affiliation: Biotechnology Center, Federal University of Rio Grande do Sul (UFRGS), 91507-970 Porto Alegre, RS, Brazil.
authors.forename: Luis F
authors.initials: LF
authors.lastname: Revers
authors.affiliation: Biotechnology Center, Federal University of Rio Grande do Sul (UFRGS), 91507-970 Porto Alegre, RS, Brazil.
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Cardone
authors.affiliation: Biotechnology Center, Federal University of Rio Grande do Sul (UFRGS), 91507-970 Porto Alegre, RS, Brazil; Federal University of Health Sciences of Porto Alegre (UFCSPA), 90050-170 Porto Alegre, RS, Brazil.
authors.forename: Bruna F
authors.initials: BF
authors.lastname: Immich
authors.affiliation: Federal University of Health Sciences of Porto Alegre (UFCSPA), 90050-170 Porto Alegre, RS, Brazil.
authors.forename: Dinara J
authors.initials: DJ
authors.lastname: Moura
authors.affiliation: Biotechnology Center, Federal University of Rio Grande do Sul (UFRGS), 91507-970 Porto Alegre, RS, Brazil; Department of Biophysics, Federal University of Rio Grande do Sul (UFRGS), 91507-970 Porto Alegre, RS, Brazil.
authors.forename: Temenouga N
authors.initials: TN
authors.lastname: Guecheva
authors.affiliation: Biotechnology Center, Federal University of Rio Grande do Sul (UFRGS), 91507-970 Porto Alegre, RS, Brazil.
authors.forename: Diego
authors.initials: D
authors.lastname: Bonatto
authors.affiliation: Biotechnology Institute, University of Caxias do Sul (UCS), 95070-560 Caxias do Sul, RS, Brazil.
authors.forename: Jomar P
authors.initials: JP
authors.lastname: Laurino
authors.affiliation: Federal University of Health Sciences of Porto Alegre (UFCSPA), 90050-170 Porto Alegre, RS, Brazil.
authors.forename: Jenifer
authors.initials: J
authors.lastname: Saffi
authors.affiliation: Department of Biological Sciences, State University of Santa Cruz (UESC), 45662-900 Ilhéus, BA, Brazil.
authors.forename: Martin
authors.initials: M
authors.lastname: Brendel
authors.affiliation: Biotechnology Center, Federal University of Rio Grande do Sul (UFRGS), 91507-970 Porto Alegre, RS, Brazil; Department of Biophysics, Federal University of Rio Grande do Sul (UFRGS), 91507-970 Porto Alegre, RS, Brazil; Biotechnology Institute, University of Caxias do Sul (UCS), 95070-560 Caxias do Sul, RS, Brazil. Electronic address: pegas@cbiot.ufrgs.br.
authors.forename: João A P
authors.initials: JA
authors.lastname: Henriques
mesh_terms: D003432:Cross-Linking Reagents / Q000494:pharmacology; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000235:genetics*; D008730:Methoxsalen / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D051721:Rad52 DNA Repair and Recombination Protein / Q000235:genetics; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D020798:Two-Hybrid System Techniques; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; C086930:RAD50 protein, S cerevisiae; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; C086929:XRS2 protein, S cerevisiae; C094326:PRK1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D004706:Endodeoxyribonucleases; C090162:PSO2 protein, S cerevisiae; D008730:Methoxsalen
keywords: DNA repair; Interstrand cross-link; MRX complex; PSO2/SNM1; SAK1/PAK1; Two Hybrid System