title: Interaction of Ddc1 and RPA with single-stranded/double-stranded DNA junctions in yeast whole cell extracts: Proteolytic degradation of the large subunit of replication protein A in ddc1Δ strains.
abstract: To characterize proteins that interact with single-stranded/double-stranded (ss/ds) DNA junctions in whole cell free extracts of Saccharomyces cerevisiae, we used [(32)P]-labeled photoreactive partial DNA duplexes containing a 3'-ss/ds-junction (3'-junction) or a 5'-ss/ds-junction (5'-junction). Identification of labeled proteins was achieved by MALDI-TOF mass spectrometry peptide mass fingerprinting and genetic analysis. In wild-type extract, one of the components of the Ddc1-Rad17-Mec3 complex, Ddc1, was found to be preferentially photocrosslinked at a 3'-junction. On the other hand, RPAp70, the large subunit of the replication protein A (RPA), was the predominant crosslinking product at a 5'-junction. Interestingly, ddc1Δ extracts did not display photocrosslinking of RPAp70 at a 5'-junction. The results show that RPAp70 crosslinked to DNA with a 5'-junction is subject to limited proteolysis in ddc1Δ extracts, whereas it is stable in WT, rad17Δ, mec3Δ and mec1Δ extracts. The degradation of the RPAp70-DNA adduct in ddc1Δ extract is strongly reduced in the presence of the proteasome inhibitor MG 132. We also addressed the question of the stability of free RPA, using anti-RPA antibodies. The results show that RPAp70 is also subject to proteolysis without photocrosslinking to DNA upon incubation in ddc1Δ extract. The data point to a novel property of Ddc1, modulating the turnover of DNA binding proteins such as RPAp70 by the proteasome.
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine Siberian Division of the Russian Academy of Sciences, Prospect Lavrentieva 8, Novosibirsk 630090, Russia; Novosibirsk State University, Novosibirsk 630090, Russia.
authors.forename: Maria V
authors.initials: MV
authors.lastname: Sukhanova
authors.affiliation: Centre National de la Recherche Scientifique, UPR4301, Centre de Biophysique Moléculaire, 45071 Orléans Cedex 02, France.
authors.forename: Claudine
authors.initials: C
authors.lastname: D'Herin
authors.affiliation: Centre National de la Recherche Scientifique, UPR4301, Centre de Biophysique Moléculaire, 45071 Orléans Cedex 02, France. Electronic address: serge.boiteux@cnrs-orleans.fr.
authors.forename: Serge
authors.initials: S
authors.lastname: Boiteux
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine Siberian Division of the Russian Academy of Sciences, Prospect Lavrentieva 8, Novosibirsk 630090, Russia; Novosibirsk State University, Novosibirsk 630090, Russia. Electronic address: lavrik@niboch.nsc.ru.
authors.forename: Olga I
authors.initials: OI
authors.lastname: Lavrik
mesh_terms: D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D059748:Proteolysis; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C440013:Ddc1 protein, S cerevisiae; C099938:MEC3 protein, S cerevisiae; D009687:Nuclear Proteins; D021122:Protein Subunits; C501395:RAD17 protein, S cerevisiae; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins
keywords: Ddc1 checkpoint protein; Double-stranded/single-stranded DNA junctions; Photoaffinity labeling; Proteasome; Protein degradation; Replication protein A (RPA)