Recombination and Repair
In recent years, a clear link has been established between genome instability and cancer. The research in our laboratory is focused on understanding how cells use recombination processes to promote chromosome stability and thereby allow the faithful reproduction of the genome. In particular we want to understand how chromosomal breaks are repaired by homologous recombination.
In eukaryotes, genetic recombination occurs in germ-line cells at meiosis where it provides a mechanism for the exchange and reassortment of genetic information. Recombination is also important in somatic cells for the repair of damaged or broken chromosomes. Defects in recombination result in unrepaired chromosomal breaks or aberrant gene translocations, the consequences of which can be catastrophic to the cell and lead to cancer. Our interest in the contribution of genetic recombination to the repair of DNA double-strand breaks stems from observations indicating that recombination-deficient cells exhibit a spontaneous genome instability phenotype coupled with ionising radiation sensitivity. Of particular interest is the discovery that cell lines derived from patients predisposed to breast cancer through mutations in BRCA1 and BRCA2 exhibit phenotypic properties characteristic of a recombination/repair defect. Thus, the genome instability associated with mutations in the BRCA genes, caused by defective recombination processes, appears to underlie certain inheritable breast and ovarian cancers.
Three-dimensional structure of a Holliday junction, a central intermediate in homologous recombination.
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Genome stability related to BRCA2 defects
Our main interest how homologous recombination repairs double-strand breaks during S-G2 phase of the cell cycle when the chromosomes have undergone replication continues to progress well. We know that efficient recombinational repair requires a number of proteins including RAD51, RAD52, RAD54, the RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3), BRCA1 and BRCA2. We have been successful in purifying many of these proteins and biochemical studies to determine their DNA substrate specificities are in progress.
Of particular importance in recombinational repair is RAD51, a homolog of the bacterial RecA protein, which catalyses the key reactions required for DNA pairing and strand exchange. In response to DNA damage RAD51 localises to distinct sub-nuclear assemblies, known as foci, where the repair reactions are thought to take place. The localisation of RAD51 to repair foci is dependent upon the breast cancer-associated tumour suppressor BRCA2, with which RAD51 interacts. The current hypothesis is that BRCA2 controls RAD51 activity. Individuals carrying mutations in BRCA1 or BRCA2 are predisposed to breast and/or ovarian cancers. Approximately, 20% of breast cancers are inheritable, and of these about one third have been linked to mutations in BRCA1 or BRCA2. In the United Kingdom alone, there are approximately 80,000 predisposed individuals of whom more than 70% will develop cancer. One defective copy of BRCA1 or BRCA2 in the genome is sufficient to confer cancer predisposition, and the loss of the second allele is commonly observed in tumour cells isolated from predisposed individuals. BRCA2 is a very large protein (mass 384 kDa) that contains a series of eight degenerate motifs, of which six have been shown to bind RAD51. These motifs, known as the BRC repeats, are approximately 30 amino acids long and are interspersed along a 1200 amino acid central region of the protein encoded by exon 11. Additionally, there is an unrelated RAD51 binding site located at the C-terminus of BRCA2. Studies in the mouse have shown that deletion of the very C-terminal region (exon 27) of BRCA2 confers a classical BRCA2 recombination- and repair-deficient phenotype and affects the localisation of RAD51 to repair foci in response to DNA damage, despite the fact that all the BRC repeats remained intact. Recently, we found that the RAD51 interaction domain at the C-terminus of BRCA2 contains a site (Serine 3291) that is phosphorylated during the cell cycle by cyclin-dependent kinases, or is dephosphorylated in response to DNA damage. Remarkably, phosphorylation of S3291 appears to act as a switch that regulates interactions of this region with RAD51. Activation of this site is therefore a critical modulator of the recombinational repair pathway (Esashi et al., Nature 434, 598-604 [2005]).
DNA repair defects and neurodegenerative diseases
Recently, our work defined the molecular defect associated with a neurological disorder known as Ataxia with Oculomotor Apraxia-1 (AOA1). We found that the product of the Aptx gene, Aprataxin, which is defective in individuals with AOA1, acts as a proofreader for abortive DNA ligation reactions (Ahel et al., Nature 443, 713-716 [2006]) . All cells, especially neuronal cells, are subjected to high levels of oxidative stress resulting in the formation of DNA strand breaks. When these breaks are repaired by a DNA ligase, it is not uncommon for the reaction to stall at an intermediate stage, such that ‘abortive ligation intermediates’ accumulate. It was found that Aprataxin specifically interacts with these intermediates and removes the AMP residue that ligase leaves covalently bound to the 5’-side of the unrepaired nick after abortive ligation. The loss of Aprataxin activity is particularly evident in neurological tissue that, due to its non-proliferative nature, is unable to utilize alternative (replication-associated homologous recombinational repair) mechanisms to remove these lesions. Without Aprataxin, unrepaired nicks with 5’-AMP moieties will accumulate in the neuronal tissue over a period of years until they are sufficiently numerous to pose a significant block to transcription. Many neuronal cells will then undergo apoptosis and cell death leading to the characteristic neurological features associated with the disorder AOA1. Thus, Aprataxin plays a critically important cellular role in guarding the genome against DNA damages that would otherwise pose a block to normal cellular processes, illustrating the link between defective DNA repair and human disease.
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Resolution of Holliday junctions during genetic recombination and DNA repair
Homologous recombination can lead to the formation of structures in which interacting DNAs become joined at four-way junctions, or Holliday structures, and these intermediates need to be resolved to allow proper chromosome segregation. All domains of life feature specialized nucleases, known as the Holliday junction resolvases, which specifically recognize junctions and resolve them by the introduction of symmetrically related nicks close to the crossover point to produce nicked duplex products. Because the resolvases introduce cuts with perfect symmetry, the nicks in the products can be sealed by DNA ligase without need for further processing. Resolvases have been isolated from a variety of organisms including bacteriophage (T4 endonuclease VII, T7 endonuclease I), bacteria (E. coli RuvC), yeast (ScCce1, SpYdc2, although these are mitochondrial resolvases), and archaea (Hje, Hjc). Surprisingly, these nucleases bear little similarity to each other at the level of amino acid sequence, so it has not been easy to identify eukaryotic orthologs. However, a mammalian activity that fits the resolvase paradigm was first detected in 1990, partially purified and given the name ResA. Recently, the fractionation of HeLa extracts, coupled with a TAP-tagged library screen for resolvase activities in S. cerevisiae, was used to identify ResA as the product of the human GEN1 gene (YEN1 in S. cerevisiae). This identification of GEN1/Yen1 as bone fide Holliday junction resolvases represents the culmination of 18 years of work (Ip et al., Nature 456, 357-361 [2008]).
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