Transcription-coupled removal of formaldehyde-induced DNA-protein crosslinks

Abstract

DNA-protein crosslinks (DPCs) form following exposure to various alkylating agents including environmental carcinogens, cancer chemotherapeutics, and reactive aldehydes. If not repaired, DPCs can interfere with key biological processes such as transcription and replication and activate programmed cell death. A growing body of evidence implicates nucleotide excision repair (NER), homologous recombination, and other mechanisms in the removal of DPCs. However, the effects of genomic context on DPC formation and removal have not been comprehensively addressed. Using a combination of next generation sequencing and DPC enrichment via protein precipitation, I showed that, unlike spontaneous DPCs, formaldehyde-induced DPCs are non-randomly distributed across the human genome, based on chromatin state. I also showed that the efficiency of DPC removal correlates with transcription at loci transcribed by RNA polymerase II. Using repair mutant cell lines, I found that efficient removal of chromosomal DPCs requires both the Cockayne syndrome group B gene as well as ‘downstream’ transcription-coupled-NER factor xeroderma pigmentosum group A gene. In contrast, I found that loci transcribed by RNA polymerase I showed no evidence of transcription-coupled DPC removal. Finally, using pharmacological inhibition of Rad5, I was able to show a reduced efficiency of DPC removal. Taken together, the results indicate that complex interactions between chromatin organization, transcriptional activity, and numerous DNA repair pathways dictate genomic patterns of DPC formation and removal.