Replication fork stability and fork restart

Lead Research Organisation: University of Sussex
Department Name: Sch of Life Sciences

Abstract

Our DNA encodes all the characteristics of our bodies. It is a library of information containing all the instructions to make a person. Each of the millions of cells in our bodies contains two complete copies of this library written on 46 DNA molecules. This DNA must be duplicated (replicated) every time one of our cells divides. DNA replication is a very complex process that ?photocopies? several billion letters of information in a few hours. DNA replication is fraught with difficulties and the replication machinery that achieves this feat is frequently disturbed by barriers to progress that are often associated with the DNA itself. The consequence of not accurately replicating the DNA in one of our cells is a change to the DNA sequence. This means a change to the information in the library. If a number of such changes accumulate, this can reprogram a cell to grow when it should not be growing. Such uncontrolled cell growth is the basis of all cancers. It is therefore important to understand how cells respond when the replication machinery stops at a barrier.

Work using organisms that grow as single cells, and are thus relatively simple, has identified many ways that cells stabilize replication machines when they are arrested at barriers. It has also identified ways in which cells can restart these arrested replication machines. Importantly, while organisms that grow as single cells are relatively simple, they use very similar ways of dealing with replication barriers as human cells do. In this program of work I therefore plan to use one of these simple organisms to study the detailed mechanism by which the DNA replication machinery is stabilized when it encounters a replication barrier. I also propose to study how these replication machines are restarted when the barrier is removed or finally overcome.

Importantly, a feature of cancer cells is that they grow more than other cells in our body and thus they replicate their DNA more often. Because of this, many cancer treatments actually deliberately introduced barriers to DNA replication in order to selectively kill cancer cells. We hope that, by understanding how the replication machinery tolerates such barriers, we may be able to increase the efficiency of cancer treatments by finding ways of making cancer cells even more likely to be killed by drugs that impede DNA replication.

Technical Summary

The DNA in our cells is at its most vulnerable when it is being replicated. A significant proportion of the mutations, chromosomal rearrangements and epigenetic changes associated with human genetic disease and cancer are thought to be generated by stochastic and DNA damage-induced replication errors. Furthermore, many chemotherapeutic drugs are specifically toxic to replicating cells, a feature that imparts therapeutic index but which also limits their utility due to side effects on rapidly dividing normal cells.
The DNA Damage Response (DDR) pathways act to suppress genetic change in the face of endogenous and exogenous replicative stress. It has become evident that the majority of cancer cells are defective in one or more DDRs. Some DDRs, such as the intra-S phase checkpoint, are proposed to be mutated in order for a pre-cancerous cell to overcome the barrier to carcinogenesis imposed by oncogene-induced senescence. Mutations in other DDRs are proposed to be selected for because they allow increased genome stability, thus promoting tumour development and evolution.
My long-term aim is to provide detailed mechanistic information that will assist the development of rational ?synthetic lethality? therapies in order to target cancers with specific changes or combinations of changes in DDR proteins and pathways, as well as to help identify potential new targets for drug development. Therefore, in this program I propose to study the relationship between DNA replication and the DDR to investigate how these pathways contribute to maintaining genome stability. Specifically, I will use a model eukaryote, S. pombe, to help to elucidate and understand how the intra-S phase checkpoint regulates DNA metabolism to maintain replication forks in a stable conformation. I will also explore the mechanisms and consequences of replication fork restart, which is necessary when these stabilisation pathways fail.
I hypothesise that a relatively under-explored protein, TopBP1, plays important roles in coordinating the transitions made by the replication and repair apparatus during fork arrest and restart by virtue of its ability to act as a multi-functional phospho-protein binding scaffold: TopBP1 can respond to a range of regulatory phosphorylation events and thus act as a ?node? coordinating signalling pathways by integrating disparate phosphorylation events. I will therefore continue my exploration of TopBP1 functions in yeast and higher eukaryotes.

Publications

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Daigaku Y (2015) A global profile of replicative polymerase usage. in Nature structural & molecular biology

 
Description MRC Training and Career Strategic advisory group
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Wellcome Investigator
Amount £2,000,000 (GBP)
Funding ID 110047/Z/15/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2016 
End 04/2021
 
Title Single molecule motion blurring 
Description A methodology to measure in vivo chromatin association 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact Research publications 
 
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 2013 Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact A successful open day with several hundred visitors, including a special tour for teachewrs

Public engagement
Year(s) Of Engagement Activity 2013
 
Description Brighton Science Festival 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Participate in Science week. Presentation of genetics to children for example. 10+ scientists involved each year

Informing public
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010,2011,2012,2013,2014
 
Description Debate Judge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact Debate. 40-60 students attending, 4 Schools represented

Inspire Youngsters
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014
 
Description School Talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Two 1 hour talsk on biology, 200 pupils 2 scholls attending. 2 scientists

inspiring youngsters
Year(s) Of Engagement Activity 2008
 
Description Work Experience 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Orgainise 1 week lab experience for 4-6 students, participants labs from across the Centre

Inspiring youngsters
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010,2011,2012,2013,2014