Replication of DNA damage, and the role of the SMC5-6 protein complex in the responses to DNA damage

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

Abstract

The genetic information, which determines the precise functions of every cell in the body, is contained in the sequence of the three billion nucleotide bases that make up the DNA molecules in the 46 chromosomes of every human cell. Our DNA is being continually damaged by exposure to carcinogenic chemicals and sunlight. Fortunately, we have evolved a whole series of DNA repair and other damage response mechanisms, to enable us to withstand almost every kind of DNA damage and thereby maintain the integrity of the genome.

We know that DNA repair is important for human health because there are several genetic diseases that are caused by defects in one or other of these DNA repair processes. Individuals with xeroderma pigmentosum (XP) are unable to deal with damage inflicted in DNA by ultraviolet light from the sun. As a consequence, they develop skin cancers at a frequency some 1000-fold above that in the general population.

Before a cell divides, it must replicate its DNA precisely, i.e. all of the three billion bases must be correctly copied. When DNA is damaged, most of the damage is repaired, but repair can be quite slow, and a rapidly growing cell is faced with the problem of having to replicate damaged DNA. Enzymes that replicate DNA are called DNA polymerases, and there is a recently discovered class of these DNA polymerases, which have special properties that enable them to replicate DNA containing damage. Whereas the majority of XP patients simply cannot remove damage from their DNA, a substantial minority, known as XP variants, is deficient in one of these special polymerases. The aim of this proposal is to understand how these special polymerases work inside the cell, what happens in XP variant cells when one of the polymerases is absent, and what the broader implications are for carcinogenesis in general. These special polymerases might prove to be suitable targets for chemotherapeutic drugs, and understanding how they work will assist in rational drug design.

The SMC proteins are a family of proteins that maintain the structure of the chromosomes, during the many complicated transactions that occur in the cell. One of these family members is also involved in repairing DNA damage and we are trying to understand how these proteins work.

Technical Summary

Replication past unrepaired damage utilises a complex series of mechanisms in eukaryotic cells. Translesion synthesis (TLS) is a major pathway for replicating past DNA damage and is effected by the Y-family of DNA polymerases (pols). Pol eta is required to replicate past UV damage and its absence in xeroderma pigmentosum variants results in very high frequencies of sunlight-induced skin cancers. The polymerase sliding clamp PCNA is mono-ubiquitinated when the replication fork stalls at DNA damaged sites and this increases its affinity for Y-family pol eta, providing a model for polymerase switching. We will (1) dissect the regulation of PCNA ubiquitination and de-ubiquitination; (2) generate forms of PCNA that cannot be ubiquitinated or that are permanently ubiquitinated and examine the effects of these alterations on the responses to DNA damage; (3) microdissect the events at the stalled replication forks, using in vitro systems and fluorescent lifetime imaging; (4) analyse the biological significance of our recent collaborative discovery of ubiquitinated forms of pol eta and iota; (3) determine which proteins are involved in a back-up process that deals with UV damage in the absence of pol eta. We have shown that Y-family pol kappa is unexpectedly involved in nucleotide excision repair. We will identify the precise role of pol kappa in NER and examine its ubiquitination and stability.

The Smc5-6 complex is involved in several responses to DNA damage. Four non-SMC proteins are associated with Smc5 and 6 in the complex from S. pombe. We will (1) examine the ubiquitination activity of the complex and its interaction with DNA; (2) identify components of the mammalian complex and other interacting proteins; (3) analyse the biological functions of the complex using mutant mice and siRNA methodology.

Publications

10 25 50
 
Description Research Grant
Amount £487,238 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 06/2010 
End 06/2013
 
Title Disorder mutations 
Description Mutation spectra for genetic disorder 
Type Of Material Database/Collection of Data/Biological Samples 
Year Produced 2006 
Provided To Others? Yes  
Impact Understanding genotype-phenotype relationshsips 
 
Title Polymerase cell lines 
Description Cell lines expressing normal or mutant forms of Y-family DNA polymerases 
Type Of Material Cell line 
Year Produced 2008 
Provided To Others? Yes  
Impact Widely used in research in different labs 
 
Description Exonuclease I 
Organisation University of Milan
Department Biomolecular Science and Biotechnology
Country Italy 
Sector Academic/University 
PI Contribution We hosted the student in my lab where she carried out crucial experiments using our expertise
Collaborator Contribution They carried out most of the research. The student whose project this work was spent an EMBO Frllowship in my klab for some collaborative experiments using our expertise
Impact Paper published in PNAS
Start Year 2010
 
Description Pol kappa and UDS assays 
Organisation Nagasaki University
Country Japan 
Sector Academic/University 
PI Contribution Joint research project between our labs
Collaborator Contribution Joint research project between our labs
Impact 19179371
Start Year 2008
 
Description Research Collaboration Ogi 
Organisation Nagasaki University
Department Research Centre for Genomic Instability and Carcinogenesis
Country Japan 
Sector Academic/University 
PI Contribution Provided well characterised cells from patients, and discussion with collaborator as well as helping to write up PMID: 22466610
Collaborator Contribution Much of the experimental work was done by partners.
Impact PMID: 22466610
Start Year 2009
 
Description Translesion synthesis in S pombe 
Organisation National Center for Scientific Research (Centre National de la Recherche Scientifique CNRS)
Department Laboratory of Genome Instability and Carcinogenesis
Country France 
Sector Academic/University 
PI Contribution Joint research project between our labs
Collaborator Contribution This was a joint research project between our labs
Impact 20453833 20452294
Start Year 2007
 
Description Ubiquitination and translesion synthesis 
Organisation Goethe University Frankfurt
Country Germany 
Sector Academic/University 
PI Contribution Both labs carried out the design, execution anmd writing up of the work.
Collaborator Contribution Joint research project between our labs
Impact 20159558
 
Description Ubiquitination of PCNA 
Organisation Unilever
Country United Kingdom 
Sector Private 
PI Contribution Research student funded on this collaboration carried out the work
Impact 19221475 18845679
 
Title Diagnostic tests 
Description We have developed diagnostic tests for three rare disorders 
Type Diagnostic Tool - Non-Imaging
Current Stage Of Development Small-scale adoption
Year Development Stage Completed 2006
Development Status Under active development/distribution
Impact Tests have assisted clinicians in diagnosis and prognosis, enabled prenatal diagnoses. Early detection has enabled preventative measures to be taken