Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8320774 | DNA Repair | 2014 | 11 Pages |
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 3â²-ss/ds-junction (3â²-junction) or a 5â²-ss/ds-junction (5â²-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 3â²-junction. On the other hand, RPAp70, the large subunit of the replication protein A (RPA), was the predominant crosslinking product at a 5â²-junction. Interestingly, ddc1Î extracts did not display photocrosslinking of RPAp70 at a 5â²-junction. The results show that RPAp70 crosslinked to DNA with a 5â²-junction is subject to limited proteolysis in ddc1Î extracts, whereas it is stable in WT, rad17Î, mec3Î and mec1Î extracts. The degradation of the RPAp70-DNA adduct in ddc1Î 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 ddc1Î extract. The data point to a novel property of Ddc1, modulating the turnover of DNA binding proteins such as RPAp70 by the proteasome.
Related Topics
Life Sciences
Biochemistry, Genetics and Molecular Biology
Biochemistry
Authors
Maria V. Sukhanova, Claudine D'Herin, Serge Boiteux, Olga I. Lavrik,