Final Report Summary - DNAREPAIR (Defects in DNA strand break repair and links to inheritable disease)
Our genetic material (DNA) is continually subjected to damage, either from endogenous sources such as reactive oxygen species produced as by-products of oxidative metabolism, from the breakdown of replication forks during cell growth, or by agents in the environment such as ionising radiation or carcinogenic chemicals. To cope with such damage, cells employ elaborate and effective repair processes that are each specialised to recognise different types of lesions in DNA. These repair systems are essential for the maintenance of genome integrity. Some individuals, however, are genetically predisposed to crippling diseases or cancers that are the direct result of mutations in genes involved in the DNA damage response.
For several years we have been interested in the mechanisms of homologous recombination, how they contribute to the repair of DNA double-strand breaks, and how they promote genome stability. The process of homologous recombination (HR) requires a number of proteins including RAD51, RAD52, RAD54, the RAD51 ‘paralogs’ (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3), BRCA2, PALB2 and RP-A. Many of these proteins have been purified in this laboratory, and we use biochemical, cytological and molecular biological approaches to understand how they function within the cell to repair DNA breaks. Since women carrying BRCA2 mutations have a 70% chance of developing breast cancer, we are determined to understand the precise role of the BRCA2 tumour suppressor in DNA repair mediated by recombination. Towards this goal, we recently succeeded in purifying the BRCA2 protein from human cells. Using biochemical analyses and electron microscopic visualisation techniques, we found that the protein binds specifically to single-stranded DNA (ssDNA), or to regions of ssDNA present at replication-fork structures. In collaboration with Prof Xiaodong Zhang’s group at Imperial College London, we were successful in determining the three-dimentional structure of BRCA2 and proposed a model for how BRCA2 facilitates RAD51 nucleoprotien filament formation, in reactions that are essential for the initiation of recombinational repair and tumour avoidance.
Individuals with Bloom’s Syndrome (BS) suffer from a genetic disorder that leads to dwarfism, immunodeficiency and reduced fertility. BS patients also develop various types of cancers, often at a young age. Cells derived from individuals with BS exhibit an extreme form of genome instability, the hallmark feature of which is an elevated frequency of sister chromatid exchanges. Bloom’s syndrome is caused by mutations in the BLM gene, which encodes BLM helicase, a protein that forms a complex with topoisomerase III and the RMI1 and RMI2 proteins. We have purified this complex, known as the BTR complex, and are currently determining its structure and mechanism of action. The BTR complex plays an important role in the resolution of joint molecules that arise through recombination. However, in addition to the BTR complex, there are two other mechanisms for the processing of joint molecules that involve the MUS81-EME1 and GEN1 endonucleases. We found that inactivation of MUS81 and GEN1 from cells derived from Bloom’s syndrome patients led to an unusual aberrant chromosome morphology and cell death. Our analysis showed that the BTR complex normally resolves joint molecules in a manner that specifically avoids sister chromatid exchanges (and loss of heterozygosity when recombination occurs between homologous chromosomes rather than sister chromatids), and that, in the absence of BTR, joint molecule resolution is mediated by the two nucleolytic pathways for resolution requiring MUS81-EME1 or GEN1. Use of these alternatives allows the cell to separate recombining chromosomes, but also comes at a heavy price since BS cells exhibit genome instability and patients suffer a broad range of early onset cancers.
Knowing that cells possess three distinct mechanisms for the resolution of joint molecules left us with a puzzle – how is it that mitotic cells use the BTR complex for joint molecule resolution rather than MUS81-EME1 or GEN1? Conversely, how is it that meiotic cells preferentially use the MUS81-EME1 and GEN1 pathways to promote chromosome segregation and form the crossovers necessary for the bipolar orientation and segregation of our maternally and paternally inherited homologous chromosomes? Mitotic and meiotic cells appear to possess similar pathways of resolution, but the way that they are used or regulated is clearly different. Our work led us to show that the specialized chromosome segregation patterns of meiosis and mitosis, which require the coordination of recombination with cell cycle progression, are achieved by regulating the timing of activation of the two crossover-promoting endonucleases. In yeast meiosis, we discovered that Mus81-Mms4 (the ortholog of MUS81-EME1) and Yen1 (the equivalent of GEN1) are controlled by phosphorylation events that modulate their activities throughout the cell cycle. Mus81-Mms4 was hyper-activated by Cdc5-mediated phosphorylation in meiosis I, in order to generate the crossovers necessary for chromosome segregation. In contrast, Yen1 was activated in meiosis II, where it catalyses the resolution of persistent Holliday junctions that would otherwise block chromosome segregation. In both yeast and human mitotic cells, similar regulatory networks are thought to restrain both nuclease activities until mitosis, biasing the outcome of recombination towards non-crossover products while also ensuring the elimination of any persistent joint molecules. Mitotic regulation of these nucleases thereby facilitates chromosome segregation while limiting the potential for loss of heterozygosity and sister-chromatid exchanges.
In addition, we investigated the cellular role of Senataxin, a protein mutated in the neurodegenerative disorder Ataxia with Oculomotor Apraxia-2. Individuals with this autosomal recessive cerebellar ataxia exhibit motor neuron degeneration, together with progressive muscle weakness and atrophy. Typically, motor coordination is affected at an early age (2 to 6 years) and progressive disability continues leading to confinement to a wheelchair in adolescent life. We found that Senataxin, which interacts directly with RNA pol II and is involved in the termination of transcription at pause sites, forms distinct nuclear foci in S/G2 phase cells. We found that the formation of these foci is dependent upon transcription and involves the formation of R-loop structures that most likely arise when replication forks collide with the transcription apparatus. Our results showed that Senataxin lies at the interface of replication stress, transcription, and the DNA damage response, and led us to propose that Ataxia with Oculomotor Apraxia is caused by defects in transcription.
For several years we have been interested in the mechanisms of homologous recombination, how they contribute to the repair of DNA double-strand breaks, and how they promote genome stability. The process of homologous recombination (HR) requires a number of proteins including RAD51, RAD52, RAD54, the RAD51 ‘paralogs’ (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3), BRCA2, PALB2 and RP-A. Many of these proteins have been purified in this laboratory, and we use biochemical, cytological and molecular biological approaches to understand how they function within the cell to repair DNA breaks. Since women carrying BRCA2 mutations have a 70% chance of developing breast cancer, we are determined to understand the precise role of the BRCA2 tumour suppressor in DNA repair mediated by recombination. Towards this goal, we recently succeeded in purifying the BRCA2 protein from human cells. Using biochemical analyses and electron microscopic visualisation techniques, we found that the protein binds specifically to single-stranded DNA (ssDNA), or to regions of ssDNA present at replication-fork structures. In collaboration with Prof Xiaodong Zhang’s group at Imperial College London, we were successful in determining the three-dimentional structure of BRCA2 and proposed a model for how BRCA2 facilitates RAD51 nucleoprotien filament formation, in reactions that are essential for the initiation of recombinational repair and tumour avoidance.
Individuals with Bloom’s Syndrome (BS) suffer from a genetic disorder that leads to dwarfism, immunodeficiency and reduced fertility. BS patients also develop various types of cancers, often at a young age. Cells derived from individuals with BS exhibit an extreme form of genome instability, the hallmark feature of which is an elevated frequency of sister chromatid exchanges. Bloom’s syndrome is caused by mutations in the BLM gene, which encodes BLM helicase, a protein that forms a complex with topoisomerase III and the RMI1 and RMI2 proteins. We have purified this complex, known as the BTR complex, and are currently determining its structure and mechanism of action. The BTR complex plays an important role in the resolution of joint molecules that arise through recombination. However, in addition to the BTR complex, there are two other mechanisms for the processing of joint molecules that involve the MUS81-EME1 and GEN1 endonucleases. We found that inactivation of MUS81 and GEN1 from cells derived from Bloom’s syndrome patients led to an unusual aberrant chromosome morphology and cell death. Our analysis showed that the BTR complex normally resolves joint molecules in a manner that specifically avoids sister chromatid exchanges (and loss of heterozygosity when recombination occurs between homologous chromosomes rather than sister chromatids), and that, in the absence of BTR, joint molecule resolution is mediated by the two nucleolytic pathways for resolution requiring MUS81-EME1 or GEN1. Use of these alternatives allows the cell to separate recombining chromosomes, but also comes at a heavy price since BS cells exhibit genome instability and patients suffer a broad range of early onset cancers.
Knowing that cells possess three distinct mechanisms for the resolution of joint molecules left us with a puzzle – how is it that mitotic cells use the BTR complex for joint molecule resolution rather than MUS81-EME1 or GEN1? Conversely, how is it that meiotic cells preferentially use the MUS81-EME1 and GEN1 pathways to promote chromosome segregation and form the crossovers necessary for the bipolar orientation and segregation of our maternally and paternally inherited homologous chromosomes? Mitotic and meiotic cells appear to possess similar pathways of resolution, but the way that they are used or regulated is clearly different. Our work led us to show that the specialized chromosome segregation patterns of meiosis and mitosis, which require the coordination of recombination with cell cycle progression, are achieved by regulating the timing of activation of the two crossover-promoting endonucleases. In yeast meiosis, we discovered that Mus81-Mms4 (the ortholog of MUS81-EME1) and Yen1 (the equivalent of GEN1) are controlled by phosphorylation events that modulate their activities throughout the cell cycle. Mus81-Mms4 was hyper-activated by Cdc5-mediated phosphorylation in meiosis I, in order to generate the crossovers necessary for chromosome segregation. In contrast, Yen1 was activated in meiosis II, where it catalyses the resolution of persistent Holliday junctions that would otherwise block chromosome segregation. In both yeast and human mitotic cells, similar regulatory networks are thought to restrain both nuclease activities until mitosis, biasing the outcome of recombination towards non-crossover products while also ensuring the elimination of any persistent joint molecules. Mitotic regulation of these nucleases thereby facilitates chromosome segregation while limiting the potential for loss of heterozygosity and sister-chromatid exchanges.
In addition, we investigated the cellular role of Senataxin, a protein mutated in the neurodegenerative disorder Ataxia with Oculomotor Apraxia-2. Individuals with this autosomal recessive cerebellar ataxia exhibit motor neuron degeneration, together with progressive muscle weakness and atrophy. Typically, motor coordination is affected at an early age (2 to 6 years) and progressive disability continues leading to confinement to a wheelchair in adolescent life. We found that Senataxin, which interacts directly with RNA pol II and is involved in the termination of transcription at pause sites, forms distinct nuclear foci in S/G2 phase cells. We found that the formation of these foci is dependent upon transcription and involves the formation of R-loop structures that most likely arise when replication forks collide with the transcription apparatus. Our results showed that Senataxin lies at the interface of replication stress, transcription, and the DNA damage response, and led us to propose that Ataxia with Oculomotor Apraxia is caused by defects in transcription.