Stephen West: Projects

Mammalian cells possess a large repertoire of DNA repair processes that maintain the integrity of our genetic material. Some individuals, however, 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 are linked with cancer predisposition, in particular breast cancers caused by mutation of the BRCA2 gene, acute leukemias associated with Fanconi anemia, and a wide range of cancers found in individuals with the chromosome instability disorder known as Bloom's syndrome. The primary focus of our research is to determine the molecular mechanisms of recombinational repair, and to define why defects in these processes cause cancer.

The breast cancer tumour suppressor BRCA2

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. Many of the proteins required for recombination have been purified in this laboratory, and we use biochemical, and molecular and cell biological approaches to understand how they bring about the repair of DNA breaks.

RAD51 protein, the human ortholog of the bacterial RecA protein, is responsible for the initiation of DNA strand break repair by catalysing homologous pairing and strand exchange, two reactions that are essential for recombinational repair. The RAD51 protein is targeted to DNA break sites by BRCA2, a well-known tumour suppressor.

The key role that BRCA2 plays in DNA repair is indicated by the fact that women carrying BRCA2 mutations have a 70 per cent chance of developing breast or ovarian cancers. An important aim of the laboratory is therefore to define the precise role that BRCA2 plays in recombination-directed DNA repair.

Using biochemical and structural approaches we recently determined the three-dimensional structure of BRCA2 protein, both alone and in complex with RAD51. The structure revealed that BRCA2 is a dimeric protein that interacts directly with RAD51. Two sets of RAD51 monomers are arranged on the BRCA2 dimer in readiness for the establishment of RAD51-ssDNA nucleoprotein filaments.

We find that BRCA2 overcomes the rate-limiting step of nucleoprotein filament formation by RAD51, providing a molecular basis for the role of BRCA2 in the maintenance of genome stability and suggesting why mutations in this protein lead to tumourigenesis.

Figure 1

Figure 1: Three-dimensional image reconstructions of the BRCA2-RAD51 complex (shown in side and top views). The BRCA2 subunits are shown in cyan and yellow, and four RAD51 monomers arranged in a semi-helical arrangement are indicated in red. (Click to view larger image)

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

Recombinational repair requires a reciprocal exchange of DNA strands between sister chromatids or homologous chromosomes, leading to the formation of DNA intermediates, such as Holliday junctions (HJs), in which the two interacting DNAs become covently interlinked.

The efficient processing of these joint molecules is essential for chromosome segregation at cell division, and is also important for determining the outcome of recombination: for example, crossovers (COs) between homologous chromosomes are required for meiotic division, whereas non-crossovers (NCOs) are favoured in mitotic cells in order to avoid 'loss of heterozygosity' (LOH), a known driver of tumourigenesis.

A four-protein complex, made up from the BLM helicase, topoisomerase IIIα, RMI1 and RMI2 (the BTR complex) promotes the dissolution of HJs to form non-crossovers. Individuals with mutations in the BLM helicase suffer from a genetic disorder called Bloom's Syndrome (BS) 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 (SCEs). We have purified this complex, known as the BTR complex, and are currently determining its structure and mechanism of action.

The high frequency of SCEs observed in cells derived from individuals with BS arise from the use of different pathways for HJ processing that involve the MUS81-EME1, SLX1-SLX4 and GEN1 endonucleases. Recent work has shown that the nucleolytic resolution of recombination intermediates is coordinated with cell cycle progression, and is restricted to the late stages of the cell cycle.

Using yeast as a model system, we discovered that the HJ-resolving activities of 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. In mitotic cells, Mus81-Mms4 is hyper-activated by Cdc5-mediated phosphorylation at G2/M, whereas Yen1 is activated at anaphase, where it catalyses the resolution of persistent Holliday junctions that would otherwise block chromosome segregation. Similar regulatory systems exist in human cells, where MUS81-EME1 is activated by CDK/PLK1 phosphorylation at prometaphase. In this case, however, the phosphorylation of MUS81-EME1 stimulates its interaction with a second structure selective endonuclease known as SLX1-SLX4, to form the MUS-SLX complex. With respect to GEN1, we found that this HJ resolvase is predominantly cytoplasmic in interphase, and only gains access to the DNA when the nuclear membrane breaks down. These two waves of Holliday junction resolution, which occur at prometaphase and mitosis, respectively, process persistent HJs and ensure chromosome segregation (Figure 2).

Figure 2

Figure 2: Recombination intermediates are resolved by three distinct mechanisms that ensure chromosome segregation. (Click to view larger image)

These three pathways for HJ resolution are essential for cell viability. Inactivation of MUS81 and GEN1 from cells derived from individuals with Bloom's syndrome leads to an unusual aberrant chromosome morphology and cell death. It appears that the BTR complex normally resolves joint molecules in a manner that specifically avoids SCEs (and loss of heterozygosity when recombination occurs between homologous chromosomes rather than sister chromatids), and that, without BTR, joint molecule resolution is mediated by MUS81-EME1-SLX1-SLX4 and GEN1. However, the importance of these nucleases is demonstrates by observations showing that cells suffer severe chromosome abnormalities and show signs of mitotic catastrophe, such as anaphase bridges (Figure 3), lagging chromosomes, and a high frequency of multi-nucleated cells.

Figure 3

Figure 3. Loss of MUS-SLX and/or GEN1 resolvase activity allows recombination intermediates to persist to mitosis where they cause defects in chromosome segregation that leads to severe genome instability. This image shows defects in chromosome segregation, indicated by the presence of anaphase bridges.

Senataxin and the maintenance of genome stability in neuronal cells

Neuronal tissues are exposed to high levels of oxidative stress and have low levels of antioxidant enzymes, making them particularly sensitive to defects in DNA repair. Due to their limited capacity for self-renewal, unrepaired lesions can accumulate over a period of years, potentially blocking transcription by RNA polymerase II (RNAPII), and trigger cell death either by progressively depriving the cell of vital transcripts or through apoptosis. As a consequence, genome instability in the adult brain leads to impaired neural development and neurodegeneration.

We are interested in understanding the molecular defects that lead to the neurodegenerative syndromes collectively known as Ataxia with Oculomotor Apraxia (AOA). These syndromes are subdivided into 3 sub-classes: AOA1 is caused by mutations in APTX, AOA2 by mutations in SETX and the chromosomal defect in AOA3 is presently unknown. Other neurological diseases, including Amyotrophic Lateral Sclerosis 4 (ALS4), Tremor-Ataxia Syndrome (TAS), Autosomal Dominant proximal Spinal Muscular Atrophy (ADSMA) and Charcot-Marie-Tooth (CMT) disease have also been linked with mutations in the SETX gene.

Previously, we discovered that the product of the APTX gene, Aprataxin, is a novel DNA-repair protein that resolves abortive DNA ligation intermediates by catalysing the removal of 5'-DNA adenylates that form when DNA ligases attempt to rejoin 'dirty' breaks caused by oxidative DNA damage. The protein also acts upon non-ligatable RNA-DNA junctions.

We are now determining the cellular role of Senataxin and why mutations in SETX cause AOA2. The product of the SETX gene, Senataxin, is a 2,677 amino acid protein that contains an RNA/DNA helicase superfamily I domain. Although the molecular functions of Senataxin, and how mutations therein lead to neuropathy remain unknown, this putative RNA/DNA helicase is considered to be an important player in the resolution of RNA/DNA hybrids (R-loops) formed during transcription termination or the RNA-DNA damage response (RDDR).

Previous studies have shown that SETX localizes to sites of collision between components of the replisome and the transcription apparatus and that it is targeted to R-loops, where it plays an important role at the interface of transcription and RDDR. Accumulating evidence indicates that R-loops may be an important source of replication stress-induced tumourigenesis. We find that loss of SETX in both human and mouse cells causes hypersensitivity to treatment with agents that cause either replication stress or induce the formation of R-loops. Furthermore, SETX deficiency promotes the formation of replication stress-induced genomic instability and chromosomal rearrangements.

We are now using genomic approaches to determine whether loss of SETX results in altered gene expression, differential methylation patterns or copy number alterations at replication stress hotspots.

Stephen West
+44 (0)20 379 62071

  • Qualifications and History
  • 1977 PhD in Biochemistry, Newcastle University, UK
  • 1978 Research Associate, Department of Therapeutic Radiology, Yale University, USA
  • 1985 Established lab at the Imperial Cancer Research Fund, UK (in 2002 the Imperial Cancer Research Fund became Cancer Research UK)
  • 2015 Group Leader, the Francis Crick Institute, London, UK