Structure-function analysis of transcription-associated DNA repair

Summary

Environmental pollutants, radiation and cellular metabolites have the propensity to damage DNA and give rise to serious health threats, including genome instability and age-related diseases. The versatile Nucleotide Excision Repair (NER) pathway is an important defence mechanism which removes a broad spectrum of DNA-helix destabilizing lesions induced by e.g., solar UV-light and certain chemicals. Transcription-blocking DNA lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which is essential to protect against DNA damage-induced cellular toxicity, safeguard genome stability and preserve transcription programs. Mutated TC-NER factors are associated with severe neurodegeneration and premature aging. TC-NER is initiated by lesion-stalled RNA polymerase II, which, subsequently, triggers the assembly of several crucial TC-NER-specific proteins, including CSB, CSA and UVSSA. Each of these proteins reside in different protein complexes with (partly) known molecular functions, however, their composition and possible modifications are unknown, as well as how these protein-complexes interact and collectively control and drive progression of TC-NER. Our main objective is to unveil their dynamic (time-resolved) assembly and structure to provide mechanistic understanding of this essential biological process. To understand the extraordinarily complexity of TC-NER, a multi-disciplinary approach using cutting-edge technologies is required: starting from the analysis of atomic structures and molecular interactions (cross-linking mass-spectrometry (XL-MS), cryo-electron microscopy (cryo-EM) and macromolecular x-ray crystallography (MX)), through in vivo functional experiments (live cell image analyses and functional assays). XL-MS has been shown to be particularly powerful in combination with cryo-EM to provide detailed insight into structures of macromolecular complexes involved in relevant biological processes. Our goal is to obtain a comprehensive map of changes of DNA damage-induced protein interactions and their perturbation as a result of mutations linked to pathological conditions, which will provide the framework for detailed functional follow-up experiments using live cell imaging analyses and functional assays. Our promising preliminary data support the feasibility of this study and the high potential to obtain important mechanistic insight into TC-NER and, consequently, to understand the functional significance of TC-NER to counteract general DNA damage-induced diseases.

Details

Project number

714.017.003

Main applicant

Prof. W.V. Vermeulen

Affiliated with

Erasmus Universiteit Rotterdam, Erasmus MC, Genetica

Duration

01/10/2017 to 30/09/2021