Presentation on theme: "Understanding the DNA Holliday Junction Benjamin C. Gale, James H. Thorpe, Nick H. Hopcroft and Christine J. Cardin. The School of Chemistry, University."— Presentation transcript:
Understanding the DNA Holliday Junction Benjamin C. Gale, James H. Thorpe, Nick H. Hopcroft and Christine J. Cardin. The School of Chemistry, University of Reading. Competitive dialysis or competition dialysis was initially described by Ren and Chaires 1 and has since then been adapted in this laboratory. The aim of such an experiment intercalate into some of the oligonucleotide sequences and further displace the osmosis equilibrium so that more drug will pass from the beaker to the Crystallographic Studies Homologous recombination is characterised by the formation of branched DNA molecules called Holliday Junctions. The role of metal ions in solution is key to the HJ. The crystal structures reveal the binding of metal ions to the HJ. Small differences gives rise to quite dramatic effects which could be important to junction recognition. p53 Consensus Sequence The tumour suppressor protein p53 is mutated in over 50% of all human cancers. The p53 consensus sequence defines the DNA sequence elements with which p53 interacts. The half site of this sequence has a striking similarity to the HJ forming sequences. The sequence d(GGGCTAGCCC) will be characterised to see if it adopts the HJ conformation. Competitive Dialysis This experiment will seek to determine which sequence a drug preferentially binds to in solution. This will allow crystallographic studies to be focussed on the most relevant sequences and drugs. The method is based on simple equilibrium dialysis. A range of drugs are available for study including those that have sub-nanomolar IC 50 values in tumour cell lines and have entered Phase 1 clinical trials. Acknowledgements For funding I thank the EPSRC, the BBSRC and the Association for International Cancer Research. Thanks also to Xenova, and in particular to Peter Charlton for supplying many of the drugs being used. For useful correspondence on the competition dialysis method I would like to thank Jonathan Chaires. Finally I would like to thank the many colleagues who have helped me including staff at DESY in Hamburg. Homologous recombination (HR) is key for life, acting to create genetic diversity and to repair double strand breaks in DNA. However the key role now appears to be in the repair and resetting of DNA replication forks that have stalled or collapsed at sites of DNA damage 1. Either way HR This work has investigated two sequences, d(TCGGTACCGA) and d(CCGGTACCGG) in the presence of Ca 2+ and Sr 2+. In the presence of Sr 2+, d(TCGGTACCGA) forms the half-X structure with a spine of five Sr 2+ sites that spiral down each B-DNA arm. By contrast in the presence The pivotal role of the tumour suppressor protein p53 in cancer is well documented with over 50% of all cancers having associated with them, mutations in p53 1,2. Previous work has identified the p53 consensus sequence, which defines the DNA sequence elements with which p53 interacts 3. ~565 base pair sequence, this represents a high level of specificity. This study will look to characterise the half site 5'- PuPuPuC(A/T)(T/A)GPyPyPy-3’ of the p53 consensus Crystallographic StudiesCompetitive Dialysis p53 Consensus SequenceFundamental Points is characterised by the formation of branched DNA molecules called Holliday Junctions (HJ’s) 2. The structures of the DNA HJ’s solved in this group have provided strong evidence of features now associated with this motif. In particular the requirement of the central d(ACC) core and quite specific stabilising contacts have been shown to be key 3,4,5,6. Until recently however 7,8 the role of metal ions in the crystal structure of the HJ has been limited despite their dramatic effect in solution 9. X-ray crystallography has revealed how one core domain, the central half of p53 binds to a quarter site 4 and further a model has been proposed in which four identical domains can occupy all four quarter isites in the full consensus sequence though this has never been seen in an X-ray crystallography experiment 4. Further to this it has been demonstrated through NMR that human p53 can bind to the Holliday junction 5. In this work it was demonstrated that 80-96% of p53 were specifically located at the junction with only 4% at the ends. For an of Ca 2+, only two ion sites have been refined in the short arms, removed from the terminal bases. For the second sequence, four Ca 2+ ion sites can be located. Two are in the long arms, found removed from the terminal bases whereby in the short arms the two sites are found at the terminus of the sequence causing the junction to be closed up. Preliminary results for Sr 2+ indicate two ion sites, one in the short arm at the terminus and one in the long arm removed from the terminal bases. sequence, which has a striking similarity to the HJ forming sequences to see if it is pre- disposed to being a HJ or if the binding of p53 or a range of anti- cancer drugs effects the conformation adopted. Crystallisation studies are currently underway for the sequence d(GGGCTAGCCC), which has been initially screened with the Hampton Research Nucleic Acid screen to yield small microcrystals. Optimised conditions are now being utilised and it is hoped that suitable crystals will be available for data collection in the later half of this year. is to determine which sequence a drug preferentially binds to in solution and hence provides great insight into the drugs that should be crystallised with specific sequences. The method works by equilibrium dialysis. A macromolecule is paced inside a semi-permeable membrane, called a dispodialyser, that have pore sizes to prevent escape of the macromolecule but allow the surrounding drug solution to enter. The dialysers are then left to equilibrate in a beaker containing a stirred solution of drug, which can cross the membrane of the dispodialysers and bind to the most preferred DNA sequence. More technically the drug in the beaker will first enter the dispodialysers by simple osmosis, to equilibrate the pressure difference between both sides of the membranes of the dialysers. The drug will then dispodialysers to compensate for the binding to DNA. Osmotic pressure and intercalation will therefore start to compete up to a point when equilibrium is reached. The amount of drug up taken by each sequence is compared by UV-visible spectroscopy after equilibrating overnight. This work has initially focussed on adopting the protocol described by Ren and Chaires to suit our requirements. Though there has been only limited success to date it is expected that this technique will provide the rationalisation for crystallisation studies within this research area. A range of drugs are available for use with this study including XR5944, which has been shown to have sub-nanomolar IC 50 values in tumour cell lines and entered Phase 1 clinical trials in the UK in July 2003. 1 McGlynn, P. et al, PNAS, 2001. 98: p. 8227-8234. 2 Holliday, R., Genet. Res., 1964. 5: p. 282-304. 3 Eichman, B. F. et al, PNAS, 2000. 97: p. 3971-3976. 4 Ho, P.S. et al, Curr. Opin. Struct. Biol. 2001. 11: p. 302-308. 5 Ortiz-Lombardia, M. et al, Nat. Struct. Biol. 1999 6: p. 913-917. 6 Thorpe, J.H. et al Acta Cryst, 2002. D58: p. 567-569. 7 Thorpe, J.H. et al, J. Mol. Biol, 2003. 327: p. 97-109. 8 Vargason, J.M. et al, J. Biol. 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