Stephen West : Genetic Recombination

Mammalian cells possess a large repertoire of DNA repair processes that maintain the integrity of our genetic material. But some individuals carry mutations in genes required for DNA repair, and this often leads to inheritable disease. An important repair process involves recombination, and defects in this process have been linked with cancer predisposition, in particular breast cancers caused by mutation of the BRCA2 gene, acute leukaemias associated with Fanconi Anemia, and a wide range of cancers found in individuals with the chromosome instability disorder known as Bloom’s syndrome. The focus of our research is to determine the molecular mechanisms of recombination, and to define why defects in these processes cause cancers.

Our genetic material – DNA – is continuallysubjected 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.

The BRCA2 tumour suppressor

For several years we have been interested in mechanisms of homologous recombination, how they contribute to the repair of DNA double-strand breaks, and thereby 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 lab, 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 (Thorslund et al., 2010; Nat Struct Mol Biol. 17: 1263-1265). 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. Importantly, BRCA2 interacts directly with the RAD51 recombinase and directs RAD51 to bind to ssDNA at sites where DNA repair reactions are initiated. Without BRCA2, RAD51 fails to localise to sites of DNA damage, providing a molecular basis for the role of BRCA2 in the maintenance of genome stability and suggests why mutations in this protein lead to tumourigenesis. Current work is focussed on determining the three-dimensional structure of BRCA2 protein and will define how BRCA2 facilitates the formation of the RAD51 nucleoprotein filaments that initiate recombinational repair.

Genome instability disorders linked to defects in the resolution of recombination intermediates

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 enzymatic properties.

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 (Wechsler et al.,2011; Nature. 471: 642-646). 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.

Regulation of nucleases that determine our genetic make-up

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 quite different.

Figure 1 Model for the timing and control of joint molecule resolution in mitotic and meiotic cells. In mitotic cells, the endonuclease activities of the two crossover promoting endonucleases MUS81-EME1 (Mus81-Mms4 in yeast) and Gen1 (Yen1 in yeast) are restrained until late in the cell cycle. This control mechanism ensures that most joint molecules are resolved into non-crossover products (thus avoiding loss of heterozygosity and sister chromatid exchanges). In contrast, in meiosis, Mus81 endonuclease is activated in a timely fashion in order to form the crossovers necessary for proper chromosome segregation, with Yen1 providing a ‘safeguard’ function.

Our work led us to show that the specialised 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 (Matos, Blanco et al., 2011; Cell. 147: 158-172). 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, a similar regulatory network was found 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 of heterozygosity and sisterchromatid exchanges.