DNA-Protein Cross-links: Formation in Cells and Tissues, Repair, and Inhibition of DNA Transcription

Abstract

DNA is constantly damaged by exogenous and endogenous agents, generating a range of nucleobase lesions. It is important to understand the biological consequences and repair mechanisms of DNA adducts. Cellular proteins can become covalently trapped on DNA to generate DNA-protein crosslinks (DPCs). Because of their unusually bulky nature, DPCs are anticipated to block many cellular processes including replication, transcription, and repair. However, cellular effects of DPCs have not been fully elucidated. Chapter 1 of this thesis provides background information on the formation, biological consequences, and repair pathways of DPCs studied in previous studies. In Chapter 2, we employed a quantitative nanoLC-ESI+-MS/MS assay to investigate the formation of free radical-induced DPCs between thymidine in DNA and tyrosine sidechains of proteins. This methodology was used to examine the role of SPRTN protease and immunoproteasome in DPC repair in human cells and mouse models. In Chapter 3, a mass spectrometry based CTAB assay was used to study the effects of DNA-peptide crosslinks on transcription in human cells. We constructed plasmid molecules containing DPCs between C5 of dC and lysine sidechains of polypeptides in order to mimic conjugates that form endogenously at DNA epigenetic marks (5-formylc-dC). Lesion bearing and control plasmids were transfected into human cells, and the amounts of RNA transcripts were determined using a mass spectrometry based approach. Moreover, DNA lesion bearing plasmid models were used to determine the importance of NER pathway in DPC repair. In Chapter 4, we investigated in vivo formation of DPCs in cells exposed to monofunctional alkylating agent, methyl methanesulfonate (MMS). A mass spectrometry-based TMT proteomics approach was used to characterize MMS-induced DNA-protein cross-linking in Chinese hamster lung fibroblasts (V79). utilizing Our results revealed that DPCs can be produced via nucleophilic attack of proteins at the C8 position of N7-methylguanine (MdG). Our results revealed novel DPC formation mechanisms and the toxicities of monofunctional agent induced DPCs. In summary, mass spectrometry-based quantification was used to the amounts of free radical induced DPCs in cells, providing evidence for the role of DPC proteolysis in repair, while CTAB assay demonstrated the effect of endogenously formed DPCs on transcription. Moreover, a mass spectrometry-based methodology was applied to examine a novel DPC formation mechanism following treatment with monofunctional alkylating agents.