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Molecular Modelling of Damaged DNA complexed with DNA Repair ProteinsAuthor: Kartheek Date: 2019-03-30 Report no: IIIT/TH/2019/16 Advisor:Marimuthu Krishnan AbstractDNA is the storehouse of genetic information in biological cells. It plays a vital role in transmission of the stored hereditary information from one generation to the next via the gene expression process. DNA damages caused by various damaging agents including free radicals, UV radiation, and anticancer drugs can alter genetic information and adversely affect gene expression pathways leading to various complex genetic disorders and cancers. To safeguard genetic information, biological cells have sophisticated survival mechanisms to recognize and rectify DNA damages with high fidelity. The task of recognition and repair of damaged DNA is carried out by a group of specific proteins. It is essential to understand the molecular basis of protein-mediated DNA damage repair to comprehend on the initiation and evolution of various complex genetic diseases and disorders. A critical molecular event that occurs during most protein-mediated DNA repair processes is the extrusion of orphaned bases at the damaged site facilitated by specific repairing enzymes. The molecular-level understanding of the mechanism, dynamics, and energetics of base extrusion is necessary to elucidate the molecular basis of protein-mediated DNA damage repair. The present thesis investigates the molecular mechanism and energetics of protein-induced base flipping and of DNA bending in various damaged DNA-protein complexes using molecular dynamics simulation and enhanced sampling free energy methods. In particular, we have explored the molecular mechanism of dinucleotide extrusion in a mismatched DNA (containing a stretch of three contiguous thymidine-thymidine base pairs) facilitated by Radiation sensitive 4 (RAD4), a key DNA repair protein, on an atom-by-atom basis using molecular dynamics (MD) and umbrella-sampling (US) simulations. Using atomistic models of RAD4-free and RAD4-bound mismatched DNA, the free energy profiles associated with extrusion of mismatched partner bases are determined for both systems. The mismatched bases adopted the most stable intra-helical conformation and their extrusion was unfavorable in RAD4-free mismatched DNA due to the presence of prohibitively high barriers (> 12.0 kcal/mol) along the extrusion pathways. Upon binding of RAD4 to the DNA, the global free energy minimum is shifted to the extra-helical state indicating the key role of RAD4-DNA interactions in catalyzing the dinucleotide base extrusion in the DNA-RAD4 complex. The critical residues of RAD4 contributing to the conformational stability of the mismatched bases are identified and the energetics of insertion of a -hairpin of RAD4 into the DNA duplex is examined.The conformational landscape-based mechanistic insight into RAD4-mediated base extrusion provided here may serve as a useful baseline to understand the molecular basis of xeroderma pigmentosum C (XPC)-mediated DNA damage repair in humans. In order to understand the molecular basis of why RAD4 recognizes certain mismatched DNA sequences with higher specificity, we have examined the base flipping dynamics in different RAD4-DNA complexes with different mismatched DNA sequences. The sequence dependent variations in the activation barrier for base extrusion and the most-stable conformations of mismatched partner bases are examined. The simulation results reveal that the energetics of base flipping and stability of the damaged bases are sensitive to the sequence of the damaged DNA. We have also explored the molecular basis of DNA damage recognition by enzyme 8-oxoguanine DNA glycosylase (MutM) and examined the base flipping energetics of guanine (G) and 8-oxoguanine (8OG) in the presence and absence of enzyme MutM. A detailed analysis of free energy profiles have revealed atomistic insights into enzyme-binding induced base flipping energetics, pathways and barriers for all the four systems. Similarly, to understand the sequence dependent damage (8OG)-induced distortions, the free energies of base flipping and DNA bending are calculated for 32 different DNA sequences. Our simulations revealed that base flipping is highly sequence dependent. Depending on the position of nucleotides on either (3' and 5') side of the base, significant alterations have been observed in base flipping and DNA bending energetics. Full thesis: pdf More details Centre for Computational Natural Sciences and Bioinformatics |
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