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Structural investigation of DNA recombination in Gram-positive organisms

Reference	BB/E017576/1
Principal Investigator / Supervisor	Dr John Rafferty
Co-Investigators / Co-Supervisors
Institution	University of Sheffield
Department	Molecular Biology and Biotechnology
Funding type	Research
Value (£)	271,725
Status	Completed
Type	Research Grant
Start date	01/01/2008
End date	31/12/2010
Duration	36 months

Abstract

The selection, manipulation and cleavage of branched DNA substrates are critical processes in all cells. The four-way DNA branchpoint known as a Holliday junction is the key intermediate in homologous recombination. We wish to study the structures and some biophysical characteristics of a set of proteins from a Gram-positive prokaryotic background that bind Holliday junctions. The data will be correlated with biochemical assays and genetic information in order to understand better how they select Holliday junctions, alter their conformation, target or deter the binding of other proteins and in some cases cleave the DNA at specific base sequences. To these ends we would seek to determine the structures by X-ray crystallography of : i) B. subtilis RecU Holliday junction resolvase bound to a DNA Holliday junction ii) B. subtilis RuvA Holliday junction branch migration machinery component iii) Lactococcus lactis phage bIL67 RuvC resolvase and its complex with a DNA junction iv) B. subtilis LrpC protein with a DNA Holliday junction Furthermore we would seek to carry out surface plasmon resonance experiments on all four proteins and DNA junction substrates either individually or in complex.

Summary

Homologous DNA recombination is a fundamental process in all living things including humans and involves the exchange of sections of DNA between two regions DNA that are very similar to each other. By swapping sections of the DNA, damage can be repaired and hence the genetic code maintained or alternatively some diversity generated. As part of the process, a crossover point called a Holliday junction is formed between the DNA regions that links them together. How the Holliday junction is manipulated into particular conformations and subsequently cut in order to release the two regions of DNA from each other again is of critical importance in deciding what sort of products are formed by the recombination. We are interested in studying proteins that especially bind to Holliday junctions. They can change its shape, help other proteins bind and/or cut it at particular places. We hope to help explain how some of these proteins recognize Holliday junctions selectively and carry out their various functions. To do this we are intending to determine the structures of the proteins when they are bound to the DNA. Specifically we want to study the proteins RuvA, RecU, RuvC and LrpC from a class of bacteria, some of which are known to cause serious infections in humans. We will also compare and contrast this vital information on the binding of Holliday junctions with that from other organisms including humans to see what common features exist.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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