Award details

Nucleosome positioning factors and DNA double-strand breaks

Reference	BB/F012810/1
Principal Investigator / Supervisor	Dr Nicholas Kent
Co-Investigators / Co-Supervisors
Institution	Cardiff University
Department	School of Biosciences
Funding type	Research
Value (£)	332,584
Status	Completed
Type	Research Grant
Start date	09/06/2008
End date	08/06/2011
Duration	36 months

Abstract

Chromosomal DNA double-strand breaks (DSBs) are created accidentally by certain genotoxins or ionising radiation and also deliberately during normal cellular processes which recombine genetic information. I have been using powerful time-resolved nucleosome mapping methods and reverse genetics to characterise chromatin remodelling events at a naturally induced DSB within the yeast MAT locus which is created by the HO endonuclease. I have recently shown that two different modules of the conserved RSC ATPase complex function to position nucleosomes at MAT. One module acts after DSB induction to promote subsequent repair, the other acts before DSB induction occurs to promote access for HO. Chromatin remodelling ATPases therefore play profound roles in both eukaryotic DSB repair and the regulation of programmed genetic exchanges. This project will use a similar strategy to test whether modules of RSC or other chromatin remodelling factors are required for two further novel nucleosome remodelling events at HO-induced DSBs. Chromatin immunopreciptiation experiments will be used to corroborate factor binding predictions and to define functional roles for remodelling events. Homologous MAT sequences also exist at the HML and HMR loci but are rendered inaccessible to HO cleavage via heterochromatin formation. The second part of this project will compare nucleosome positions at MAT and HM loci, together with ChIP of RSC subunits, in mutants affecting heterochromatin formation and histone acetylation. This will test the hypothesis that recruitment and/or activity of RSC at the un-cleaved MAT locus is mediated by specific histone tail modifications. This project will yield novel insights into the mechanisms of DSB repair and the regulation of programmed recombination events thus aiding our understanding of alterations and failures in human DSB processing which contribute to the ageing process and disease development.

Summary

DNA BREAKS AND DISEASE: The long helical DNA molecules which encode genetic information in chromosomes are not indestructible. DNA molecules can be damaged, and in the most extreme cases completely severed. These breaks are actually quite common and occur either accidentally due to the action of chemicals or radiation, or deliberately when cells cut their own DNA molecules as part of natural processes which shuffle genetic information. Whatever the origin of the break, a cell will die if the severed ends of DNA are not rejoined because broken fragments of chromosome can be cast adrift when a cell divides. Although cells possess multiple systems to recognise and repair broken DNA, these mechanisms are not perfect and may be disrupted in certain diseases. A decline in overall repair effectiveness may also contribute to ageing. In the worst case, mis-joining DNA molecules can scramble genetic information and reprogram cells to become cancerous. My lab is therefore interested in how break formation and repair works and, ultimately, whether we can manipulate the process as part of possible disease therapies. DNA BREAKS ARE CHROMATIN BREAKS: DNA in chromosomes is wrapped together with proteins to form a complex called chromatin. The proteins in chromatin act as packaging and insulation for DNA and every process occurring in the cell nucleus including the damage and repair of DNA occurs in the context of chromatin. We and others have recently discovered that manipulation of chromatin proteins is critical to efficient repair of DNA breaks. We have devised a technique to observe chromatin changes during the creation of DNA breaks in yeast cells, and discovered that a factor called RSC is required, immediately after break creation, to move chromatin proteins away from the chromosome ends in order for DNA repair enzymes to access the cleaved DNA. This process is analogous to an electrician peeling back the insulation at the end of a wire in order to make a connection. PROJECT OBJECTIVES: Objectives 1 and 2: Not only have we been examining chromatin just as breaks occur, we have also been following its structure during subsequent repair. Just after the DNA molecules are repaired, we can observe a process in which chromatin is also restored to its normal state. During this part of the project, we will use a similar strategy to the one we employed to discover RSC to understand whether or not this resetting process is important for repair and to identify specific factors responsible. Objective 3: Much of the system we study in yeast is based on a naturally induced chromosome break in a region called MAT. Repair of the MAT break is used to swap genetic information in a manner which allows yeast to change sex. A similar process occurs in human cells when genes are shuffled before eggs and sperm are produced and during the generation of antibodies to fight disease. We have been able to show that an alternative form of RSC in yeast is required for MAT breakage. Different forms of RSC therefore promote both DNA breakage and its subsequent repair. We will test the idea that the different forms of RSC recognise their targets through certain molecular tags added to chromatin. These experiments will tell us how chromatin may be used to regulate natural chromosomal breakage processes. Objective 4: Although much of our work centres on the MAT system in yeast, we have also been examining breaks which take place elsewhere. Some changes to chromatin proteins surrounding other broken DNA ends still occur in cells lacking RSC, suggesting that another early-acting chromatin-remodelling factor may be required for effective DNA repair. We will therefore test cells with genetic defects in chromatin remodelling and DNA repair to find the factor which is responsible.

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	Microbiology
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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