Research
Introduction
The metazoan genome is large, complex and contains many repetitive sequences. These factors make it a considerable challenge for cells to accurately replicate and segregate chromosomal DNA. Without accurate DNA copying and separation mechanisms, organismal development, growth and tissue regeneration are severely perturbed. Higher organisms have therefore evolved unique pathways that help them to maintain and reproduce their complex genomes. A striking example of the importance of these pathways comes from my own postdoctoral work on the function of the vertebrate BRCA2 gene product. It was observed that mutation that truncates this crucial protein precipitates catastrophic chromosome breakage in dividing vertebrate cells. The broad aim of my research programme is to identify additional pathways that like BRCA2 are essential for the replication of complex genomes. Their molecular characterisation may help explain how it is possible for multicellular organisms to reproduce, grow and regenerate their tissues.
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Figure 1: Metaphase spread showing chromosomes in a mouse cell that lacks brca2. Broken and fused chromosomes can be clearly seen. Such abnormalities are rarely seen in normal cells. |
Over the last 8 years my laboratory has focused on one such pathway that came to my attention following studies on it in humans. The genetic condition Fanconi Anaemia (FA) results in a striking phenotype. Such individuals have defects in development, growth retardation, bone marrow stem cell attrition and an enormous lifetime cancer risk (1000 fold when compared to cigarette smoking which is 10 fold). It became apparent to us that FA provided a unique opportunity to dissect a complete pathway, because most of the individual components were actively being identified. What therefore needs to be established is how these molecules collude to preserve and transmit our genomes from cell to cell and generation to generation.
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Figure 2: Fanconi Anemia - the phenotype. Child with FA with growth retardation and hand abnormalities. Metaphase spread of a cell from a FA patient after exposure to DNA crosslinks shows fused chromosomes. |
The defect in FA is due to a failure to respond to and repair DNA damage during replication, but perhaps the most striking aspect concerns genetic heterogeneity: the FA phenotype can result from recessive mutations in up to 13 distinct genes. Most of these gene products appear to interact physically with each other, indicating that they are distinct components functioning in a common molecular process. Our current knowledge of the "FA pathway" can be summarized as follows. Most of the FA proteins are part of a large nuclear complex (a multiprotein E3 monoubiquitin ligase). A DNA damage trigger recruits this complex to chromatin, where it monoubiquitinates two key FA gene products called FANCD2 and FANCI. The complex, FANCD2, FANCI and the helicase FANCJ initiate the repair of DNA damage. We want to establish at a molecular level the precise nature of this repair and how these activities interact with other replication blocking DNA damage responses. Ultimately we aim to explain why this pathway is so essential for normal development, stem cell survival, and cancer suppression.
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Figure 3: Outline of the core FA pathway. DNA damage activates the FA core complex which is then ubiquitinated FANCD2 and FANCI. These 2 protiens then accumulate into dots, where they carry out repair. Little is known about the form of this repair. |
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Model systems
We use model organisms to help us dissect the FA pathway. Much of our earlier work was done using the chicken B lymphocyte cell line - DT40. This cell line is ideally suited for genetics studies because it is very easy to knock genes out in it. We have used DT40 to generate double mutants carrying defects in the FA pathway and other DNA repair proteins. This analysis allows us to determine the interaction between the FA genes and enzymes utilised in DNA replication as well as those involved in homologous recombination repair. We can also use this system to determine the function of new genes that may function in the FA pathway.
Over the last few years we have also worked on the FA pathway in the social unicellular amoeba - Dictyostelium Discoideum. We are using this organism because we think it has a much more simplified FA pathway since many of the vertebrate FA genes are missing. Dicty is also a very genetically tractable organism and we hope to devise novel screens to identify new DNA repair proteins. This work is in colaboration with Rob Kay's lab at LMB.
More recently we are making mouse knockouts in the FA pathway. Although such mice have been created by many labs and despite their phenotype being much milder than in humans, we are creating novel strains that will allow us to study cancers carrying the FA defect, as well as strains that may be prone to bone marrow failure.
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Figure 4: Evolutionary conservation of the FA pathway. Dicty may have a simplified FA pathway. The life cycle of dicty, the genome of dicty is smaller than vertabrates. For example, shown to scale is the human FANCD2 locus compared to the dicty FANCD2 locus. Although FANCD2 is highly conserved, in dicty it's ammino acids sequence contain "bizzare" poly amino acid repeats. |
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Biochemistry
Although most of the FA genes have been cloned, a striking feature is that their amino acid sequence gives us no clues to how they work in DNA repair. The FA genes are therefore known as "orphans". A significant effort in our lab concentrates on expressing and purifying FA proteins. These purified proteins are being tested for DNA repair activity. In addition to this we are trying to reconstitute the critical monoubiquitination step in the pathway in a cell free system. We collaborate with Roger Williams's lab to gain structural insight into the FA proteins by X ray crystallography.
Methods
We are developing new techniques that allow the purification of large complexes from genetically modified DT40 cells. A limitation in purifying large complexes is that they often occur at low concentrations in cells or that they may be unstable. We have devised strategies that allow us to knock in epitope tags into the genomic locus of genes coding for proteins which form in large complexes. The resulting DT40 strains now only express the relevant tagged protein under physiological control. We can then purify a tagged protein complex from the cells. Since many protein complexes are subject to control by "modifier" gene products (i.e. kinases or phosphatases) it is then possible to knock such modifiers out. This then allows the purification of complexes in different states.
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