Abstract A32: Exploring the interplay between nucleotide excision repair and DNA replicative stress

Abstract

Abstract Ultraviolet (UV) light causes highly genotoxic DNA lesions that are removed by nucleotide excision repair (NER), and NER is critical for preventing UV-associated skin cancers. UV-induced DNA lesions block the progression of DNA polymerases, leading to replicative stress and genomic instability. Upon UV, the ataxia-telangectasia and rad3 related (ATR) kinase is rapidly activated, leading to cell-cycle arrest, inhibition of replication origin firing, and stabilization of blocked replication forks. Intriguingly, previous data indicated that reduced ATR activity causes profound NER defects specifically in S phase cells. Moreover many model cancer cell lines, including a majority of melanoma lines, present striking S phase-specific NER defects. We used the yeast S. cerevisiae as model to investigate NER activity during S phase. We optimized a novel flow cytometry-based assay to quantify NER as a function of cell cycle in this organism. Using this assay we demonstrate that, as for human cells, deletion of the yeast ATR homolog Mec1 causes NER defects uniquely in S, and that initiation of DNA replication is prerequisite for manifestation of this defect. S phase-specific NER was perturbed by mutations in various genes encoding key DNA damage response factors that regulate, e.g. cell cycle checkpoints, chromatin remodeling, and homologous recombination. Our data also reveal a strong correlation between S phase-specific NER defects and elevated RPA focus formation in mutants that are sensitive to replicative stress. Finally we demonstrate that modulation of RPA levels strongly influences NER activity specifically during S in human cancer cell lines. Overall, our data indicate that mutations in a multitude of DNA damage response pathways cause inordinate sequestration of RPA at stalled replication forks, thereby reducing the availability of this factor to perform its essential role in NER. This has important implications for our understanding of UV-induced skin cancer development in humans. Citation Format: Elliot Drobetsky, François Bélanger, Jean-Philippe Angers, Émile Fortier, Santiago Costantino, Hugo Wurtele. Exploring the interplay between nucleotide excision repair and DNA replicative stress [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A32.