Exploring the Effects of Transcription Factor Binding on DNA Damage Formation and Repair at Single-Nucleotide Resolution

Abstract

Genomic studies have shown that there are elevated damage and mutation rates in the active transcription factor (TF) binding sites of several tumor types. This begs the question of how TFs interact with DNA damage to promote mutagenesis. In UV-linked cancers, this enrichment is partly attributed to the damage-susceptible conformation DNA adopts when bound by E26 transformation-specific (ETS) factors. Meanwhile, work using excision-repair sequencing datasets have revealed that enrichment of damage and mutations in active TF binding sites correlates with depleted nucleotide excision repair, suggesting that TFs may contribute to mutagenesis by binding to damaged DNA and competing with repair proteins for lesion recognition. Because DNA binding behavior differs between TF families and is sequence specific, disentangling the roles of increased damage susceptibility and repair attenuation in mutagenesis requires nuanced, TF-specific analysis. Previous research investigating the relationship between TF activity and DNA damage have primarily focused on select TFs or been done in aggregate across large cohorts of TFs at kilobase resolution. While collectively, there is evidence that TFs contribute to UV-induced mutagenesis by both enhancing initial damage formation and attenuating repair, there has yet to be a comprehensive characterization of these mechanisms on a per-TF basis. In this work, we leverage data from a several damage-mapping sequencing assays to develop a scalable, k-mer based, statistical framework that identifies and deconvolves these two mechanisms of TF-mediated mutagenesis across hundreds of TFs. Numerous previously unidentified TFs exhibit significantly differential damage formation at specific positions in their binding sites. Our survey of AlphaFold-predicted TF-DNA complexes shows that structural distortions in TF-bound DNA at positions with modulated damage formation, align with structural changes that are reported to predispose DNA to UV photodimerization, demonstrating the fundamental role of structure in UV damage formation. We also noted widespread repair attenuation in TF binding sites with a new level of resolution as to the precise TFs and binding site sequences that may be driving TF competition with repair factors. We integrated the results of this joint assessment with mutations from TF binding sites in skin cancer samples and are able to distinguish mutation peaks driven by increased damage susceptibility versus attenuated repair. This comparative analysis reveals that the mechanisms of TF-mediated mutagenesis is both TF- and position-specific and that the cooccurrence of TF-mutagenic behavior can compound mutagenesis. Throughout, we outline the key challenges of analyzing UV DNA damage and emphasize the importance of controlling for DNA sequence bias when performing these analyses in TF binding sites. Together, our work provides a robust statistical framework to elucidate the mechanisms of mutagenic TF-binding and offers novel insights into the complex interplay between protein interactions with DNA damage and repair.