Defects in replicative DNA polymerases linked to cancer predisposition and tumour development

Lead Research Organisation: University of Oxford
Department Name: Zoology

Abstract

Our genetic material is constantly subjected to damage. Much of this damage comes not from external sources such as carcinogens or radiation, but from the intrinsic biological processes that DNA has to perform to allow our cells to function. One key biological function of DNA involves its replication, so that each of the two daughter cells produced during cell division receives a complete and accurate copy of all the genetic information. Even in normal cells, this copying process is not 100% perfect, and the resulting errors can lead to mutations that alter cell function, potentially leading to cell death or other changes that can lead to cancer.

Recent findings have shown that some cancers are associated with defects in the polymerase enzymes that copy DNA. DNA polymerases are normally highly accurate, but defects in these polymerases can increase the error rate when the genome is copied, and this can accelerate the development of some types of cancer in tissues where there is a high rate of cell multiplication. In this grant we will study these defective polymerases in detail, to understand which changes found in human cells are likely to be pathogenic. We will study how the polymerase malfunctions, the consequences for the process of chromosome replication and the types of copying mistakes which result. Understanding the properties of the cancer-associated DNA polymerase should help to explain how these defects lead to tumour formation. It is also possible that drugs might be developed that target the replication of tumour cells that express these variant polymerases.

Technical Summary

Germline mutations in the exonuclease domain of replicative DNA polymerases delta and epsilon have been recently shown to be associated with a predisposition to colorectal cancer. In addition a wider range of exonuclease domain mutations have been found as somatically variants in colorectal and endometrial cancer, suggesting that changes in polymerases may more widespread as a step leading to genome instability in tumourigenesis. The exonuclease domain contains the active site for a 3'-5' exonuclease activity required for proofreading, capable of removing misincorporated nucleotides that are not correctly base paired to the template. In this grant we will characterize the polymerase variants associated with colorectal cancer predisposition and endometrial tumours by constructing equivalent mutations in a model organism, fission yeast, and by purifying human polymerases containing the relevant mutations. We will compare the phenotype of the strains constructed and the biochemical properties of the purified polymerases with exonuclease null version of the polymerases, where key catalytic residues in the exonuclease active site have been changed, to clarify the extent to which the phenotype is due to loss of exonuclease activity. We will grow strains expressing polymerases with exonuclease domain mutations for many generations and perform genomic sequencing to determine the mutational spectra and whether specific regions of the genome are prone to mutagenesis. We will establish why some exonuclease domain mutants require an intact checkpoint for survival in yeast, and whether this is relevant to the mechanism of mutagenesis. This will allow insight into the mechanism whereby polymerases with exonuclease domain defects promote tumour development in humans.

Planned Impact

The local beneficiaries of this project will be the post-doctoral research assistants working on the project who, through experience gained, will be in a better position to secure future jobs in the healthcare/research sector. The PIs' groups also have a steady flow of graduate students and MRes/undergraduate research project students who will be able to participate in aspects of the research.

The academic DNA replication and repair community will benefit from the research outputs which should clarify the consequences of human polymerase defects on S phase execution, and genome stability.

The clinical community will benefit from clarification of which human DNA polymerase mutations are likely to have a phenotype in human cells, rather than just being polymorphisms or passenger mutations, enhancing the usefulness of any genetic screening for germline mutations and possibly aiding the prognostic value of these changes for tumour classification.

The general public will benefit in that those individuals affected by polymerase mutations stand to receive better health care as a result of improved understanding of the nature of the phenotypic consequence of the mutation. This might include frequent screening for individuals at risk from germline mutations, or better prognostic information using DNA polymerase mutations in tumour classification. In the long term drugs exploiting tumour phenotypes resulting from polymerase mutations may have value for treatment of specific cancers.
 
Description EMBO short term fellowship to Dr Ignacio Soriano
Amount € 1,000 (EUR)
Organisation European Molecular Biology Organisation 
Sector Learned Society
Country European Union (EU)
Start 09/2016 
End 10/2016
 
Description EPA Cephalosporin Fund
Amount £14,400 (GBP)
Organisation University of Oxford 
Sector Academic/University
Country United Kingdom
Start 10/2018 
End 12/2018
 
Title Fission yeast mutants expressing clinical polymerase variants 
Description Fission yeast strains have been engineered to express DNA pol delta or epsilon expressing mutant variants equivalent to those found in human cells that are suspected to cause cancer predisposition. 
Type Of Material Cell line 
Provided To Others? No  
Impact In progress 
 
Title DNA replication net 
Description Website giving information on DNA replication proteins 
Type Of Material Database/Collection of data 
Year Produced 2009 
Provided To Others? Yes  
Impact Widely accessed website. 
URL http://www.dnareplication.net/
 
Description Collaboration 
Organisation University of Birmingham
Department Institute of Cancer and Genomic Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution comparison of polymerase variant phenotypes in S. pombe model organism
Collaborator Contribution Clinical data on polymerase variant phenotypes in tumours
Impact Will result in joint publications
Start Year 2017
 
Description Joint experiments 
Organisation University of Oxford
Department Nuffield Department of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution DNA polymerase purification and in vitro assays
Collaborator Contribution DNA sequencing and data analysis
Impact Will result in joint publications
Start Year 2016
 
Description Studying clinical DNA polymerase mutations 
Organisation St George's Hospital
Country United Kingdom 
Sector Hospitals 
PI Contribution Discussion of experiments
Collaborator Contribution Biochemical analysis of polymerases in vitro
Impact Ongoing experiments
Start Year 2015
 
Description Studying clinical DNA polymerase mutations 
Organisation University of Graz
Department Institute of Human Genetics
Country Austria 
Sector Academic/University 
PI Contribution Discussion of experiments
Collaborator Contribution Biochemical analysis of polymerases in vitro
Impact Ongoing experiments
Start Year 2015
 
Description Studying clinical DNA polymerase mutations 
Organisation University of Oxford
Department Wellcome Trust Centre for Human Genetics
Country United Kingdom 
Sector Academic/University 
PI Contribution Discussion of experiments
Collaborator Contribution Biochemical analysis of polymerases in vitro
Impact Ongoing experiments
Start Year 2015
 
Description Oral communication: Mutations in replicative polymerases associated with cancer predisposition 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk given at meeting of Chromosomal stability, Copenhagen October 2016
Year(s) Of Engagement Activity 2016
 
Description Poster: Mutations in replicative polymerases associated with cancer predisposition 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation at 10th 3r symposium, Matsue, Japan
Year(s) Of Engagement Activity 2016
URL http://www.3r2016.com/information.html
 
Description Talk at 12th Genome Stability Network Meeting, Robinson College, Cambridge, January 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Research talk
Year(s) Of Engagement Activity 2016
 
Description Talk at 8th International Fission Yeast Meeting Kobe Japan 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Research talk.
Year(s) Of Engagement Activity 2015
 
Description presentation: DNA polymerase proofreading-domain mutations associated with cancer 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation at 10th Quinquennial Conference on Responses to DNA damage: from molecule to disease Egmond aan Zee, The Netherlands, April 17-22, 2016
Year(s) Of Engagement Activity 2016
URL https://www.medgencentre.nl/DNA%20Repair%202016/