Exploring DNA Lesion Recognition Mechanisms for Cyclobutane Pyrimidine Dimers and 6-4 Photoproduct by Rad4

Abstract

Genome integrity is seriously threatened by UV radiation’s ability to cause damage in DNA. CPD and 6-4PP are two of the most frequent lesions brought on by Ultraviolet radiation. CPD is a DNA lesion occurring from the covalent linking of nearby pyrimidine bases through their C5 and C6 carbons, generating a cyclobutane ring. In contrast, 6-4PP develops when neighbouring pyrimidine bases in DNA become crosslinked following UV exposure, producing a covalent bond between their C6 and C4 carbons. If left untreated, these lesions can result in mutations and even the development of cancer. Clarifying the DNA repair process depends on our ability to comprehend the mechanisms underpinning DNA lesion identification. DNA damage repair proteins such as XPC/Rad4 recognize and facilitate repair of these damages. In order to better understand how XPC/Rad4 recognises DNA lesions in Nucleotide Excision Repair (NER), this thesis will focus on how it interacts with CPD and 6-4PP. The highly conserved repair process known as NER is in charge of locating and eradicating diverse DNA damages. The current thesis looks at the mechanism, energetics and dynamics of Rad4 sequence-specific lesion recognition. The flipping of a pair of partner bases that are present opposite to the lesion (CPD or 64PP) is one of the crucial biochemical steps that take place while XPC or Rad4 tries to identify and bind to a DNA lesion. Major events in NER like insertion of BHD3-β hairpin, flipping of partner bases and Rad4/XPC association with the damaged DNA were studied by formulating relevant reaction coordinates or Collective Variables. These CVs were then used in Molecular Dynamics and enhanced sampling method, Umbrella Sampling to produce energy profiles for the aforementioned events. A deeper understanding of these energy profiles reveal that insertion of BHD3-β hairpin takes place only after the flipping of partner bases has taken place and a cavity is induced in the active site of the DNA but 6-4PP containing complex promotes the insertion of BHD3-β hairpin with higher ease and flexibility compared to a CPD containing complex.This study also reveals that flipping of 5’-dA partner base takes place before flipping of 3’-dA partner base. And these observations were consistent for both lesions, CPD and 64PP. The results of this study will be useful to improve our knowledge of the molecular processes behind Rad4’s identification of DNA lesions and give light on the critical steps in the NER pathway. Finally, this information may aid in the creation of focused therapeutic approaches for preventing and treating UV-induced DNA damage, enhancing our capacity to protect genomic integrity and lessen the dangers of UV radiation exposure