Escherichia coli MutS is a DNA binding repair protein (97 kDa, monomer) and its biological significance arises from its recognition of mismatches which occur as errors during DNA replication. Mismatches and mutagenic bases represent a fascinating and diverse range of shapes and sizes and it is not obvious how a single protein (MutS) can recognise such molecular diversity against a huge background of canonical Watson-Crick base pairs. In this project, the structure of a 17mer mismatch GT DNA was carried out using NMR spectroscopy to identify the differences caused by the introduction of a non-canonical base pair on helical structure. The resulting structure was B-form in conformation and local helical distortions were observed about the GT mispair due primarily to its sheared orientation. The effect of mismatch orientation, sequence context and oligonucleotide length on mismatch stability was also investigated using UV absorbance melting and NMR spectroscopy. The results showed that substitution of a TG mispair for a GT mispair was accompanied by a small drop in melting temperature. It was also discovered that sequences in which purine-purine or pyrimidine-pyrimidine stacking occurred induced additional stability of the mismatch resulting in a higher melting temperature of the duplex.Affinity of mismatch GT DNA and its mismatch orientation, sequence context and length analogues for MutS was investigated by monitoring changes to the chemical shifts and linewidths of imino protons resonances during NMR titration. The results showed that MutS displayed higher affinity towards sequences which involved better stacking between the flanking base pairs and the GT/TG mispair.Analogous NMR structural investigations of 6-thioguanine modified 13mer GC DNA and its oxidised derivatives have been successfully carried out. The NMR structure was successfully determined of the former and the results obtained showed the effect on helical structure induced by the substitution of a different DNA lesion.Although the crystal structures of MutS bound to DNA mismatches have been known for a number of years, the analogous crystal structures of uncomplexed apo MutS have not been determined to date. Consequently, vital structural knowledge on the large change in conformation of MutS upon binding to the DNA mismatch is seriously lacking. We have successfully isolated the structurally and functionally important NTD of E. coli MutS and its labelled (13C, 15N) analogues and have shown that it is endowed with a stable, tertiary structural fold and well suited to NMR structure determination. This is exemplified by the assignments of several backbone amide and side chain resonances using isotope aided 3D NMR techniques.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:526993 |
| Date | January 2010 |
| Creators | Cheung, Tony Chun Tung |
| Contributors | Ramesh, Vasudevan |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/nmr-structural-studies-of-mismatched-dna-base-pairs-and-their-interaction-with-e-coli-muts-protein(a6441a8c-0c23-47b5-a3cb-57691e751bc1).html |
Page generated in 0.0056 seconds