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

• Published in: DNA repair Vol. 22; pp. 30 - 40
• Main Authors: Sukhanova, Maria V., D’Herin, Claudine, Boiteux, Serge, Lavrik, Olga I.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.10.2014; Elsevier
• Subjects: Cell Cycle Proteins; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Ddc1 checkpoint protein; DNA, Single-Stranded; DNA, Single-Stranded - metabolism; DNA-Binding Proteins; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Double-stranded/single-stranded DNA junctions; Life Sciences; Nuclear Proteins; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Photoaffinity labeling; Proteasome; Protein Binding; Protein degradation; Protein Subunits; Protein Subunits - metabolism; Proteolysis; Replication Protein A; Replication protein A (RPA); Replication Protein A - genetics; Replication Protein A - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Replication protein A (RPA); Double-stranded/single-stranded DNA junctions; Photoaffinity labeling; Ddc1 checkpoint protein; Protein degradation; Proteasome
• ISSN: 1568-7864; 1568-7856; 1568-7856
• DOI: 10.1016/j.dnarep.2014.07.002

•Ddc1 is crosslinked to the 3′-ss/dsDNA junction in cell extract of S. cerevisiae.•RPA is crosslinked to the 5′-ss/dsDNA junctions in cell extract of S. cerevisiae.•The large subunit of RPA (RPAp70) is proteolyzed in ddc1Δ extract.•Proteasome inhibitor, MG-132, suppresses RPAp70 cleavage in ddc1Δ extract.•Ddc1 is likely involved in regulating proteasome-dependent degradation of RPA. 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.