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 |
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Institution: | 1. Institute of Chemical Biology and Fundamental Medicine Siberian Division of the Russian Academy of Sciences, Prospect Lavrentieva 8, Novosibirsk 630090, Russia;2. Novosibirsk State University, Novosibirsk 630090, Russia;3. Centre National de la Recherche Scientifique, UPR4301, Centre de Biophysique Moléculaire, 45071 Orléans Cedex 02, France;1. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland;2. Institute of Biochemistry, Faculty of Biology, University of Warsaw, Miecznikowa 1, Warsaw, Poland;3. Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Pawińskiego 5a, Warsaw, Poland;4. Groupe «Réparation de l’ADN», CNRS UMR8200, Université Paris-Sud, Institut de Cancérologie Gustave Roussy, F-94805 Villejuif Cedex, France;1. University Hospital of Grenoble, Grenoble, France;2. Université Grenoble Alpes, Grenoble, France;3. EyeTechCare, Rillieux la Pape, France;4. Inserm, U1032, Lyon, F-69003, France;5. Université de Lyon, Lyon, France;6. Hospices Civils de Lyon, Lyon, France;1. Department of Chemistry-Biochemistry and Physics, University of Québec at Trois- Rivières C. P. 500, Trois-Rivières (Québec), G9A 5H7, Canada;2. Faculty of Medicine, Memorial University of Newfoundland, St. John''s, Newfoundland, A1B 3V6, Canada;3. College of the North Atlantic, Ridge Road Campus, Newfoundland, A1C 6L8, Canada |
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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 32P]-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. |
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Keywords: | Double-stranded/single-stranded DNA junctions Photoaffinity labeling Ddc1 checkpoint protein Replication protein A (RPA) Protein degradation Proteasome |
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