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Sukhanova, M. V., D’Herin, C., Boiteux, S. and Lavrik, O. I.

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

DNA Repair (2014) 22, 30-40 -doi : http://dx.doi.org/10.1016/j.dnarep.2014.07.002

par Frapart - publié le , mis à jour le

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.