Analyzing the associated proteome and the genomic landscape of repaired nascent chromatin after genotoxic stress with aniFOUND.

Deeper study of the UV-induced DNA Damage Responses (DDR) is crucial for the understanding of melanoma and non-melanoma skin cancers, of Nucleotide Excision Repair (NER)-related rare diseases and for the development of new chemotherapeutic approaches. Towards this, the establishment of integrative comprehensive methods to illuminate the different molecular aspects underlying UV-DDR can contribute to diagnostic and therapeutic breakthroughs. Here, we present aniFOUND (accelerated native isolation of Factors On Unscheduled Nascent DNA), an antibody-free method, which can label, capture and map post-damage, newly synthesized, repaired chromatin during NER in its native form. For a comprehensive and in depth examination of both the proteins constituting this chromatin fraction and the genome-wide distribution of the repaired DNA, we coupled aniFOUND to high-throughput analytical technologies. By mass-spectrometry we identified several proteins that are known to take part in UVC-DDR or DDR in general, as well as new repair factors with no previously known role in DDR. We experimentally validated the recruitment of two of these proteins, the chromatin remodeler RSF1 and the Cornelia de Lange Syndrome-associated factor and cohesin loader NIPBL, at sites of UVC-induced photolesions. Furthermore, coupling of aniFOUND to Next Generation Sequencing allowed us to map and characterize repair efficacy at different chromosomal regions, including at rDNA and telomeres. aniFOUND is a methodology delineating the composition and landscape of specific chromatin loci by integrating state of the art "omics" technologies to promote a comprehensive view of UVC-induced DDR.