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Title: Structural studies in DNA
Author: Booth, Ewan D.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1991
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Base pair mismatches may be formed during replication or genetic recombination. Not all mismatched base pairs are repaired with the same efficiency, in particular the A.G. mismatch is consistently under repaired. Why the A.G. mismatch is poorly repaired is not clear, although previous work has suggested the mismatch is conformationally flexible. In the present study the A.G. mismatch in the sequence dCGCAAATTGGCG was examined by X-ray crystallography and pH dependant UV melting studies. Crystals were grown at pH 6.6. The base pair has an A (anti).G(syn) conformation. From the pH dependant UV melting studies it is likely this is a protonated AH&43 (anti).G(syn) base pair formed by protonation of the N(1)- atom of adenine. The G (syn) conformation is stabilised by hydrogen bonding to a network of solvent molecules in the major and minor grooves. A parallel investigation of the A.l mismatch was performed. The oligonucleotide dCGCAAATTIGCG was crystallised. An A (anti).l(syn) base pair may be indicated, pH dependent UV melting profiles suggest the existence of a protonated base pair. 5-Bromouracile is strongly mutagenic forming mismatches with G. The G.BrU mismatch has been reported to form a wobble base pair although other forms are possible. The oligonucleotide dCGCGAATTBrUGCCG was synthesised and used in pH dependent UV melting studies. These suggest that there is little or no contribution to the base pair from non-wobble, ionised or enolised, forms. Frame shift mutations may arise from nonmatched bases in DNA. A series of tridecameras was synthesised for analysis by X-ray crystallography; dCGCGTAATTCGCG, dCGCGBrUAATTCGCG, dCGCGTAATBRUCGCG and dCGCGTAATTBrUCGCG. It was hoped to determine the looped out, stacked or nonmatched nature of the additional bases. 2-Amino-2'-deoxyadenosine (dA') is an analogue of dA. Base pairs formed between A' and T contain three hydrogen bonds and are A.T like in the major groove and G.C like in the minor groove. The additional hydrogen bonding affords extra stability to A'. T base pairs relative to A.T base pairs. Z-DNA is formed by d(GC)n sequences but not by d(AT)n sequences. The mixed sequence oligonucleotide dCA'TA'TG was made for X-ray crystallographic investigation and UV melting studies. It was hoped this would crystallise as Z-DNA. Recently X-DNA has been investigated. This can be formed by poly(dA-dT) under stringent conditions due to the effects of salt or alcohols. X-DNA was first identified by its CD spectrum. Similar CD spectra have been observed for poly(dA'-dT) closer to physiological conditions. The oligonucleotides d(TA')n n = 2,3,4 were synthesised for X-ray crystallography, d(TA')4 was also investigated by CD spectroscopy. This showed the ability of the oligonucleotide to form X-DNA. The additional stability of dA' oligonucleotides gives them potential used as stable short genetic probes. A series of oligonucleotides was made, these hybridised successfully under conditions where the native sequences failed to. A new phosporamidite for the synthesis of dA' oligonucleotides was developed. This overcomes the problem of acid catalysed depurination of dA' residues during solid phase phosphoramidite DNA synthesis, di-n-butylformamide dimethylacetal was used to protect the N6 function. This was shown to be stable to the detritylation conditions encountered in DNA synthesis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available