Regulation of genome stability by FAN1 and SLX4

Lead Research Organisation: University of Dundee

Abstract

DNA, the genetic material in our cells, is frequently damaged. Since the instructions for the proper running of the cell are contained within DNA, damage to this material poses the risk of permanent changes to these instructions, called mutations, that can have an adverse effect on cell behaviour. Mutations are the cause of cancer and other debilitating diseases. Normally, cells quickly realise when DNA has been damaged and they fix it. However, when the level of DNA damage is high or when repair is sub-optimal, the cell can't cope and launches what is known as the DNA damage response. This shuts off normal cell function to give the cell plenty of opportunity to find the damage and greatly increases the capacity to repair it. We are working on two important proteins, ATM and ATR that find DNA damage and initiate the DNA damage response. We are trying to understand how they find the damaged DNA, how they trigger the DNA damage response and what other proteins are involved. In this way, we hope to understand in detail the normal cellular processes that go wrong in cancer and how we might correct them in diseased cells, or prevent them from going wrong in the first place.

Technical Summary

Defects in DNA repair or in the ability of cells to slow cell cycle progression after DNA damage can increase cellular mutation rates and cause genome instability leading to variety of debilitating diseases that are accompanied by heightened predispostion to cancer. DNA lesions trigger a highly conserved signal transduction pathway that regulates many aspects of the cellular response to DNA damage and the principal regulators of this pathway are the ATM and ATR protein kinases, critical regulators of genome stability. These kinases rapidly translocate to sites of DNA damage or to stalled DNA replisomes where they are activated by mechanisms that are not yet clear. This causes phosphorylation of a range of proteins including the BRCA1 and p53 tumour suppressors, and the conserved Chk1 and Chk2 protein kinases that when activated by ATM/ATR in turn phosphorylate effectors of cell cycle progression. It is still not known how they carry out many of their cellular functions at the molecular level. For example, it is not clear how ATM and ATR increase cellular DNA repair capacity, how ATR prevents firing of origins of replication when the DNA replication machinery stalls, how these kinases restore chromatin configuration after DNA damage or how they promote recovery from DNA damage. The main goal of my laboratory is to understand how ATM/ATR regulate these processes. We are using several approaches biochemical, genetic and structural - to investigate how ATM/ATR recognise their substrates, what these substrates are, and how these targets contribute to the maintenance of genome stability. We are also using yeast to investigate how, at the molecular level, stalled replisomes bypass replication fork-blocking lesions.We identified several new targets of ATM and ATR and have investigated their cellular functions using budding yeast as a model organism. Phosphorylation of Esc4 by ATR, for example, is essential for the resumption of DNA replication when the DNA replication apparatus stalls at sites of DNA damage. Another new target of ATM and ATR, Slx4, prevents abnormal DNA structures from arising in the vicinity of stalled replication forks. Recently we found that Slx4 is an important regulator of the ability of stalled replisomes to bypass replisome-blocking lesions and we are currently investigating the molecular mechanisms involved. We are currently studying human Slx4-containing complexes and their role(s) in maintenance of genome stability. In mammalian cells we have shown that phosphorylation of the 53BP1 tumour suppressor at Ser25 by ATM caused 53BP1 to physically associate with the hPTIP mediator protein that we identified previously. Interaction of these two proteins is essential for intact responses to DNA damage and we are currently investigating this at the molecular level.

Publications

10 25 50
 
Description Search for Head of Research Institution
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
Impact Selection of head of prestigious UK research institution
 
Description Characterization of new anti-cancer drug targets involved in DNA repair
Amount £750,000 (GBP)
Organisation Merck 
Department Merck Serono
Sector Private
Country Germany
Start 12/2016 
End 12/2020
 
Description Chromatin and the DNA damage response
Amount £250,000 (GBP)
Organisation Marie Sklodowska-Curie Actions 
Department Initial Training Networks (ITN)
Sector Charity/Non Profit
Country Global
Start 09/2013 
End 09/2016
 
Description EMBO Lab start-up grant
Amount £300,340 (GBP)
Organisation Loulou Foundation 
Sector Private
Country Unknown
Start 01/2016 
End 12/2018
 
Description Substrates and regulation of the CDKL5 kinase
Amount £650,000 (GBP)
Organisation Loulou Foundation 
Sector Private
Country Unknown
Start 01/2016 
End 01/2019
 
Title Antibodies 
Description Antibodies against a wide variety of human and moue DNA repair proteins including XPF, ERCC1, MUS81, EME1, SLX4, FAN1 
Type Of Material Biological samples 
Year Produced 2013 
Provided To Others? Yes  
Impact Papers 
 
Title Fan1 nuclease-dead knockin mice 
Description Mice with an inactivating mutation in the Fan1 nuclease 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2016 
Provided To Others? Yes  
Impact Paper in Science which garnered world-wide attention 
 
Title Stable cell lines 
Description HEK293 and U2OS cells stably express GFP-tagged forms of a range of DNA repair proteins including DVC1, SLX4, FAN1, MUS81, XPF 
Type Of Material Cell line 
Year Produced 2013 
Provided To Others? Yes  
Impact Papers 
 
Description Analysis of kidney diseases as consequence of defective DNA repair 
Organisation Spanish National Cancer Research Center
Country Spain 
Sector Academic/University 
PI Contribution Provided mice and reagents for this project
Collaborator Contribution Generated a new mouse model of KIN, a kidney disease caused by defective DNA repair
Impact None yet
Start Year 2015
 
Description Analysis of the roles of the EXO5 nuclease in DNA repair 
Organisation University of Dundee
Department School of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Cell biological analysis of the uncharacterized nuclease EXO5 in DNA repair
Collaborator Contribution Biochemical analysis of the uncharacterized nuclease EXO5 in DNA repair
Impact None yet
Start Year 2017
 
Description Characterization of CDKL5 signalling in brain 
Organisation University of Edinburgh
Department School of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Providing data on how CDKL5 signals inside cells
Collaborator Contribution Analysis of CDKL5 signaling in brain in vivo and advanced imaging relevant to these experiments
Impact None yet
Start Year 2015
 
Description Characterization of genes mutated in novel Fanconi Anemia subtypes 
Organisation Medical University of Wurzburg
Country Germany 
Sector Academic/University 
PI Contribution Determining the mechanisms underlying the functions of novel genes mutated in Fanconi anemia and how these mechanisms are perturbed by pathological mutations
Collaborator Contribution Identifying novel genes mutated in Fanconi anemia which is caused by defective DNA repair; these genes are all new DNA repair genes
Impact Three papers
Start Year 2010
 
Description Characterization of new drugs targets involved in DNA repair 
Organisation Merck
Country Germany 
Sector Private 
PI Contribution We are helping Merck to investigate new drug targets in the DNA repair arena, and helping characterize new inhibitors of DNA repair.
Collaborator Contribution Merck provide advice and compounds and use the data generated in my lab to refine their drug programmes. The funds they provided fund three postdocs in my lab
Impact None yet
Start Year 2016
 
Description Characterization of the Exo5 DNA repair nuclease 
Organisation University of Dundee
Department MRC Protein Phosphorylation and Ubiquitylation Unit
Country United Kingdom 
Sector Public 
PI Contribution Characterization of role of Exo5 in DNA repair
Collaborator Contribution Identification of allosteric regulator of Exo5
Impact None yet
Start Year 2017
 
Description Control of stalled replication forks by the Fan1 nuclease in health and disease 
Organisation Wellcome Trust
Department Wellcome Trust Centre for Gene Regulation and Expression
Country United Kingdom 
Sector Academic/University 
PI Contribution We made cells lacking the Fan1 nuclease
Collaborator Contribution With the help of Julian Blow's lab we used tricky fibre labelling experiments to show Fan1 nuclease activity controls the speed of stalled replication forks
Impact None yet
Start Year 2014
 
Description Defining the roles of C15ORF41 in health and disease 
Organisation University of Oxford
Department Weatherall Institute of Molecular Medicine (WIMM)
Country United Kingdom 
Sector Public 
PI Contribution Cell biological analysis of the C15ORF41 nuclease mutated in CDAI-type congential anemia
Collaborator Contribution Biochemical analysis of the C15ORF41 nuclease mutated in CDAI-type congential anemia
Impact None yet
Start Year 2016
 
Description Determining how mutations in the CDKL5 kinase cause the symptoms associated with CDKL5 deficiency 
Organisation Loulou Foundation
Country Unknown 
Sector Private 
PI Contribution We are studying how CDKL5 mutations affect CDKL5 activity and substrate phosphorylation.
Collaborator Contribution Loulou Foundation keeps us in contact with other CDKL5 researchers through the CDKL5 Fourm and Portal and with patients and parents
Impact None yet
Start Year 2016
 
Description Expansion imaging of CDKL5 activity in brain 
Organisation Massachusetts Institute of Technology
Country United States 
Sector Academic/University 
PI Contribution We will provide antibodies against CDKL5 and substrates for imaging
Collaborator Contribution Our collaborator will help with revolutionary new technology he developed
Impact None yet
Start Year 2017
 
Description Functional Analysis of a novel chromatin-associated nuclease complex mutated in disease 
Organisation University of Copenhagen
Department Biotech Research and Innovation Center (BRIC)
Country Denmark 
Sector Academic/University 
PI Contribution We have used genome editing to knock out the genes encoding both subunit of the novel nuclease C15orf41-codanin to decipher the functions of this complex in maintaining genome stability
Collaborator Contribution Anja previously found interesting phenotypes in cells depleted of codanin using siRNA. We will now apply to same tests, with Anja's help, to analysing C15 function
Impact None yet
Start Year 2015
 
Description Identification of CDKL5 substrates 
Organisation Dundee Model Railway Club
Country United Kingdom 
Sector Private 
PI Contribution Follow up of the substrates of CDKL5 identified by collaborator
Collaborator Contribution Identification of CDKL5 substrates
Impact None yet - multiple outputs anticipated
Start Year 2016
 
Description Pancreatic ductal adenocarcinoma (PDAC) as a consequence of defective DNA repair 
Organisation McGill University
Country Canada 
Sector Academic/University 
PI Contribution Characterization of the effect on genome stability of mutations found in DNA repair nucleases in PDAC
Collaborator Contribution Identification of PDAC-causing mutations
Impact None yet
Start Year 2015
 
Description Pancreatic ductal adenocarcinoma (PDAC) as a consequence of defective DNA repair 
Organisation University of Toronto
Country Canada 
Sector Academic/University 
PI Contribution Characterization of the effect on genome stability of mutations found in DNA repair nucleases in PDAC
Collaborator Contribution Identification of PDAC-causing mutations
Impact Two papers
Start Year 2015
 
Description Role of the Ankle1 nuclease in maintaining genome stability 
Organisation University of Vienna
Department Max F. Perutz Laboratories (MFPL)
Country Austria 
Sector Academic/University 
PI Contribution We are crossing Ankle null mice to our Slx1 null mice to see if the synthetic lethality we observed in worms hold true in mammals
Collaborator Contribution Our collaborators made the Ankle null mice
Impact None yet
Start Year 2016
 
Description Role of the CDKL5 kinase in DNA repair and human health 
Organisation University of Sussex
Department Genome Damage and Stability Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution Biochemical analysis of CDKL5 in DNA repair and effect of pathological mutations
Collaborator Contribution Cell biological analysis of CDKL5 in DNA repair and effect of pathological mutations
Impact None yet
Start Year 2018
 
Description Strucutral and functional analysis of UBE2T in Fanconi ameina 
Organisation University of Dundee
Department MRC Protein Phosphorylation and Ubiquitylation Unit
Country United Kingdom 
Sector Public 
PI Contribution My team is analysing the impact of interesting mutations in Ube2t identified in structural analysis carried out by my collaborator
Collaborator Contribution Strutural analysis of Ube2t
Impact None yet
Start Year 2016
 
Description Acted as a judge for Debating Matters 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Acted as a judge in the local heats of Debating Matters, a nationwide debating competition for secondary school children
Year(s) Of Engagement Activity 2015,2016
 
Description Local radio interviews 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Media (as a channel to the public)
Results and Impact I was interviewed on local radio stations about our paper describing the Fan1 tumor suppressor, published in Jan 2016 in the journal Science
Year(s) Of Engagement Activity 2016
 
Description National radio interviews about recent breakthrough 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact I was interviewed on national radio stations (Sky/Radio 5 live) about our paper describing the Fan1 tumor suppressor, published in Jan 2016 in the journal Science
Year(s) Of Engagement Activity 2016
 
Description Newspaper and radio interviews 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact I was interviewed on local and national radio and by newspaper reporters about our 2016 papers on FAN1 - which we described as a Molecular Scissors preventing human disease
Year(s) Of Engagement Activity 2016
 
Description Press release 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact Local press put out an article about our paper describing the Fan1 tumor suppressor, published in Jan 2016 in the journal Science
Year(s) Of Engagement Activity 2016
 
Description Promotional video for Loulou Foundation 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Supporters
Results and Impact Promotional video as a scientist funded by Loulou
Year(s) Of Engagement Activity 2017
 
Description School visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I gave a talk to final-year high-school science students on the merits of scientific research
Year(s) Of Engagement Activity 2016
 
Description Talk at school 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talked with around 50 final year secondary school children about life as a research scientist
Year(s) Of Engagement Activity 2017