DNA damage response mechanisms

Lead Research Organisation: University of Sussex
Department Name: Brighton and Sussex Medical School

Abstract

DNA damage is caused by both internal DNA damaging agents (like oxygen) that are associated with normal life and by agents from outside our bodies such as sunlight and ionising radiation. The consequences of not repairing the DNA damage caused by these agents is the accumulation of changes in the DNA sequence of individual cells that can reprogram the cell to grow when it should not be growing. Such uncontrolled cell growth is the basis of all cancers.
It is therefore important that we understand how cells respond to DNA damage and how they repair such damage. Work using single celled (and thus relatively simple) yeast model organisms has in the past identified many DNA damage response pathways and lead to an understanding of how these function to both repair DNA damage and to prevent cells dividing when their DNA is damaged. By using these easily manipulated yeast model systems, scientists have been able to define many of the fundamental molecular mechanisms used by DNA damage response pathways and to examine these functions in the context of other cellular processes. Importantly, while yeasts are relatively simple, they use very similar ways of dealing with these problems as human cells do.
It has become clear that multiple inter-dependent DNA repair and signalling pathways act to prevent mutations occurring and thus help us avoid cancer. In this program of work I propose to study several aspects of how DNA damage response pathways operate to control the production of other proteins in the cell and thus to help the cell tolerate and repair the DNA damage. I also propose to explore how the DNA damage response mechanisms interact with the process of replicating, or copying, the DNA. Accurate DNA replication is as important as DNA repair in preventing mutations. DNA damage response pathways are known to interact with DNA replication to ensure that DNA damage does not result in miscopying (i.e. mutation). I propose to largely use the yeast model systems. However, since many of the proteins and pathways involved are found both in the yeast and in mammals, I propose to extend specific studies into mammalian cells by using the mouse model system.

Technical Summary

DNA repair and signalling pathways act to prevent genomic instability and help avoid the somatic mutations that lead to cancer. DNA-integrity checkpoint pathways detect DNA damage and/or its consequences and coordinate multiple cellular responses including cell cycle arrest, transcription and DNA repair. In addition to regulating cell cycle events, DNA damage response pathways are themselves subject to regulation by the cell cycle. For example, in different phases of the cell cycle, DNA damage response pathways are activated differently and target different downstream events. The purpose of the programme is to understand the crosstalk between DNA damage responses and the cell cycle. The experimental plans are divided into 3 sections.
1. Rad4 and its ortholog TopBP1. Rad4/TopBP1 share features of mediator proteins and function in both replication checkpoint and DNA damage checkpoint signalling. They are also required for replication initiation. Mediators are broadly defined as proteins that facilitate phosphorylation of downstream substrates by the core checkpoint kinases ATR and ATM. Mediators are generally specific to distinct DNA damage responses and different cell cycle stages. I propose that Cdk- and checkpoint pathway-dependent phosphorylation events and protein-protein interactions regulate multiple mediator functions of Rad4. I aim to test this by identifying and characterising phosphorylation sites and sites of protein-protein interaction. To move key observations from yeast to mammals we will study human and mouse TopBP1 to delineate the phenotypic consequences of mutations equivalent to those identified in yeast.
2. We recently identified two new targets of the Rad3 (ATR) checkpoint in S. pombe: transcription in unperturbed S phase and the regulation of Cullin 4 ubiquitin ligases. We will characterise these by asking: how does Rad3 contribute to Cdc10 (E2F analogue) controlled transcription in unperturbed S phase and how does it activate Cdc10-dependent transcription after DNA damage? How does the checkpoint target Cullin-4 proteolysis in G2 to establish an ?S-phase like state? conducive to DNA repair synthesis?
3. To address how DNA repair processes are regulated in S phase, we recently developed a system to monitor the outcome of site-specific replication fork stalling/collapse. We will extend this and use it to analyse checkpoint regulation of the outcome of RF stalling/collapse in different contexts including palindromic, inverted and direct repeats.

Publications

10 25 50
 
Description Carr: ERC Advanced Grant (2011)
Amount £1,972,146 (GBP)
Funding ID 268788 
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 05/2011 
End 04/2016
 
Description Carr: MRC Program Grant (2011)
Amount £1,921,176 (GBP)
Funding ID G1100074-E01/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2011 
End 09/2016
 
Title DSB repair tools for fission yeast research 
Description A system for induction and analysis of DSBs 
Type Of Material Improvements to research infrastructure 
Year Produced 2008 
Provided To Others? Yes  
Impact Publication by other research groups using tools supplied 
 
Title Transcription tools for fission yeast research 
Description A series of tools and protocols for general yeast reaearch 
Type Of Material Improvements to research infrastructure 
Year Produced 2011 
Provided To Others? Yes  
Impact Publication by other groups using the tools and reagents supplied. 
 
Description Daoshun Kong 
Organisation Peking University
Department College of Life Sciences
Country China 
Sector Academic/University 
PI Contribution Scientific collaboration. Idea, materials
Collaborator Contribution Experimental and intellectual
Impact Publication
 
Description Cancer Genetics 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Public event to introduce and discuss genetics and cancer

Nothing Specific
Year(s) Of Engagement Activity 2008
 
Description Careers Day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presnt Science at a Careers Fair. 100 + studnet

Inspire Youngsters
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010,2011,2012
 
Description Radio interview 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Comment as scientific expert on local radio

None specific
Year(s) Of Engagement Activity 2008