title: Role of DNA Repair Factor Xeroderma Pigmentosum Protein Group C in Response to Replication Stress As Revealed by DNA Fragile Site Affinity Chromatography and Quantitative Proteomics.
abstract: Replication stress (RS) fuels genomic instability and cancer development and may contribute to aging, raising the need to identify factors involved in cellular responses to such stress. Here, we present a strategy for identification of factors affecting the maintenance of common fragile sites (CFSs), which are genomic loci that are particularly sensitive to RS and suffer from increased breakage and rearrangements in tumors. A DNA probe designed to match the high flexibility island sequence typical for the commonly expressed CFS (FRA16D) was used as specific DNA affinity bait. Proteins significantly enriched at the FRA16D fragment under normal and replication stress conditions were identified using stable isotope labeling of amino acids in cell culture-based quantitative mass spectrometry. The identified proteins interacting with the FRA16D fragment included some known CFS stabilizers, thereby validating this screening approach. Among the hits from our screen so far not implicated in CFS maintenance, we chose Xeroderma pigmentosum protein group C (XPC) for further characterization. XPC is a key factor in the DNA repair pathway known as global genomic nucleotide excision repair (GG-NER), a mechanism whose several components were enriched at the FRA16D fragment in our screen. Functional experiments revealed defective checkpoint signaling and escape of DNA replication intermediates into mitosis and the next generation of XPC-depleted cells exposed to RS. Overall, our results provide insights into an unexpected biological role of XPC in response to replication stress and document the power of proteomics-based screening strategies to elucidate mechanisms of pathophysiological significance.
authors: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University , Olomouc, Czech Republic.|Department of Protein Biochemistry and Proteomics, Centre of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science, Palacky University , Olomouc, Czech Republic.
authors: Lucie
authors: L
authors: Beresova
authors: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University , Olomouc, Czech Republic.
authors: Eva
authors: E
authors: Vesela
authors: Department of Protein Biochemistry and Proteomics, Centre of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science, Palacky University , Olomouc, Czech Republic.
authors: Ivo
authors: I
authors: Chamrad
authors: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University , Olomouc, Czech Republic.
authors: Jiri
authors: J
authors: Voller
authors: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University , Olomouc, Czech Republic.
authors: Masayuki
authors: M
authors: Yamada
authors: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University , Olomouc, Czech Republic.
authors: Tomas
authors: T
authors: Furst
authors: Department of Protein Biochemistry and Proteomics, Centre of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science, Palacky University , Olomouc, Czech Republic.
authors: Rene
authors: R
authors: Lenobel
authors: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University , Olomouc, Czech Republic.
authors: Katarina
authors: K
authors: Chroma
authors: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University , Olomouc, Czech Republic.
authors: Jan
authors: J
authors: Gursky
authors: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University , Olomouc, Czech Republic.
authors: Katerina
authors: K
authors: Krizova
authors: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University , Olomouc, Czech Republic.
authors: Martin
authors: M
authors: Mistrik
authors: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University , Olomouc, Czech Republic.|Danish Cancer Society Research Center , Copenhagen, Denmark.|Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Biochemistry and Biophysics, Karolinska Institute , Stockholm, Sweden.
authors: Jiri
authors: J
authors: Bartek
mesh_terms: D059447:Cell Cycle Checkpoints; D002846:Chromatography, Affinity; D043283:Chromosome Fragile Sites; D004260:DNA Repair / Q000502:physiology*; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000502:physiology*; D006801:Humans; D040901:Proteomics / Q000379:methods*; D014983:Xeroderma Pigmentosum
chemical_list: D004268:DNA-Binding Proteins; C086984:XPC protein, human
keywords: 53BP1 bodies; DNA affinity chromatography; DNA damage response; FRA16D; SILAC proteomics; Xeroderma pigmentosum complementation group C (XPC) protein; common fragile sites; mitosis; replication stress; γH2AX