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

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.