High-resolution mapping demonstrates inhibition of DNA excision repair by transcription factors.

DNA base damage arises frequently in living cells and needs to be removed by base excision repair (BER) to prevent mutagenesis and genome instability. Both the formation and repair of base damage occur in chromatin and are conceivably affected by DNA-binding proteins such as transcription factors (TFs). However, to what extent TF binding affects base damage distribution and BER in cells is unclear. Here, we used a genome-wide damage mapping method, N-methylpurine-sequencing (NMP-seq), and characterized alkylation damage distribution and BER at TF binding sites in yeast cells treated with the alkylating agent methyl methanesulfonate (MMS). Our data show that alkylation damage formation was mainly suppressed at the binding sites of yeast TFs ARS binding factor 1 (Abf1) and rDNA enhancer binding protein 1 (Reb1), but individual hotspots with elevated damage levels were also found. Additionally, Abf1 and Reb1 binding strongly inhibits BER in vivo and in vitro, causing slow repair both within the core motif and its adjacent DNA. Repair of ultraviolet (UV) damage by nucleotide excision repair (NER) was also inhibited by TF binding. Interestingly, TF binding inhibits a larger DNA region for NER relative to BER. The observed effects are caused by the TF-DNA interaction, because damage formation and BER can be restored by depletion of Abf1 or Reb1 protein from the nucleus. Thus, our data reveal that TF binding significantly modulates alkylation base damage formation and inhibits repair by the BER pathway. The interplay between base damage formation and BER may play an important role in affecting mutation frequency in gene regulatory regions.

Transcription-coupled nucleotide excision repair: New insights revealed by genomic approaches.

Elongation of RNA polymerase II (Pol II) is affected by many factors including DNA damage. Bulky damage, such as lesions caused by ultraviolet (UV) radiation, arrests Pol II and inhibits gene transcription, and may lead to genome instability and cell death. Cells activate transcription-coupled nucleotide excision repair (TC-NER) to remove Pol II-impeding damage and allow transcription resumption. TC-NER initiation in humans is mediated by Cockayne syndrome group B (CSB) protein, which binds to the stalled Pol II and promotes assembly of the repair machinery. Given the complex nature of the TC-NER pathway and its unique function at the interface between transcription and repair, new approaches are required to gain in-depth understanding of the mechanism. Advances in genomic approaches provide an important opportunity to investigate how TC-NER is initiated upon damage-induced Pol II stalling and what factors are involved in this process. In this Review, we discuss new mechanisms of TC-NER revealed by genome-wide DNA damage mapping and new TC-NER factors identified by high-throughput screening. As TC-NER conducts strand-specific repair of mutagenic damage, we also discuss how this repair pathway causes mutational strand asymmetry in the cancer genome.

Role of Nucleotide Excision Repair in Cisplatin Resistance.

Cisplatin is a chemotherapeutic drug used for the treatment of a number of cancers. The efficacy of cisplatin relies on its binding to DNA and the induction of cytotoxic DNA damage to kill cancer cells. Cisplatin-based therapy is best known for curing testicular cancer; however, treatment of other solid tumors with cisplatin has not been as successful. Pre-clinical and clinical studies have revealed nucleotide excision repair (NER) as a major resistance mechanism against cisplatin in tumor cells. NER is a versatile DNA repair system targeting a wide range of helix-distorting DNA damage. The NER pathway consists of multiple steps, including damage recognition, pre-incision complex assembly, dual incision, and repair synthesis. NER proteins can recognize cisplatin-induced DNA damage and remove the damage from the genome, thereby neutralizing the cytotoxicity of cisplatin and causing drug resistance. Here, we review the molecular mechanism by which NER repairs cisplatin damage, focusing on the recent development of genome-wide cisplatin damage mapping methods. We also discuss how the expression and somatic mutations of key NER genes affect the response of cancer cells to cisplatin. Finally, small molecules targeting NER factors provide important tools to manipulate NER capacity in cancer cells. The status of research on these inhibitors and their implications in cancer treatment will be discussed.