Covalent trapping and structural studies of intermediates in the base excision repair pathway
DNA suffers from constant aberrant modifications in its chemical structure by both endogenous and exogenous reagents. DNA repair pathways remove such damages and restore it back to it undamaged state. Base excision repair pathway (BER) is responsible for correcting base specific damages in the genome. DNA glycosylases form the cornerstone of the BER pathway, whereby they have the dual burden of locating a damaged base in the genome and initiate the process of repair by excising it from duplex DNA.
In this thesis, we have sought to address the question of how DNA glycosylases interact with undamaged DNA. To obtain complexes of DNA glycosylases with undamaged DNA, we have successfully employed a covalent trapping technique to circumvent the problems associated with lower affinity of DNA glycosylases for undamaged DNA and formation of multiple complexes in solution. The three dimensional structures of these complexes reveal, for the first time, a high resolution description of interaction of DNA glycosylases with undamaged DNA and also lend insights about the base extrusion pathway of the damaged base.
This thesis presents undamaged DNA-bound structures of two different DNA glycosylases responsible for cleaving oxidized guanines from DNA—hOGG1, the human enzyme and MutM, the functional homolog in bacteria. In case of hOGG1, the structure reveals, for the first time, a novel extrahelical location of a flipped out guanine. Computational work performed in collaboration with Prof. Martin Karplus revealed a novel mode of dipolar interaction used by the active site of hOGG1 to discriminate between 8-oxoguanine and guanine. This structure also represents a late intermediate in the base extrusion pathway. In case of MutM, the structures reveal an early intermediate where the glycosylase is in an interrogating conformation and the DNA has all base pairs intrahelically disposed.
We have also used the same trapping strategy for obtaining the structure of the lesion-recognition complex of MutY, an adenine glycosylase in bacteria.
0490: Organic chemistry