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1.  A molecular model for the role of SYCP3 in meiotic chromosome organisation 
eLife  2014;3:e02963.
The synaptonemal complex (SC) is an evolutionarily-conserved protein assembly that holds together homologous chromosomes during prophase of the first meiotic division. Whilst essential for meiosis and fertility, the molecular structure of the SC has proved resistant to elucidation. The SC protein SYCP3 has a crucial but poorly understood role in establishing the architecture of the meiotic chromosome. Here we show that human SYCP3 forms a highly-elongated helical tetramer of 20 nm length. N-terminal sequences extending from each end of the rod-like structure bind double-stranded DNA, enabling SYCP3 to link distant sites along the sister chromatid. We further find that SYCP3 self-assembles into regular filamentous structures that resemble the known morphology of the SC lateral element. Together, our data form the basis for a model in which SYCP3 binding and assembly on meiotic chromosomes leads to their organisation into compact structures compatible with recombination and crossover formation.
eLife digest
When a sperm cell and an egg cell unite, each contributes half of the genetic material needed for the fertilised egg to develop. This creates opportunities for new and beneficial genetic combinations to arise. To ensure that each new sperm or egg has half a set of chromosomes, reproductive cells undergo a special type of division called meiosis.
During the early stages of meiosis, copies of each chromosome—one inherited from the mother, the other from the father—are paired up along the midline of the dividing cell. A protein complex known as the synaptonemal complex acts as a ‘zipper’, pulling the chromosomes in each pair closer together. The arms of the maternal chromosome and the paternal chromosome are so close that they sometimes cross over and swap a section of DNA. These crossovers perform two critical functions. First, they recombine the genetic information of a cell, so that offspring can benefit from new gene combinations. Second, they help to hold the chromosomes together at a key point of meiosis, reducing the chances that the wrong number of chromosomes ends up in a sperm or egg cell.
The zipper structure is essential for meiosis. Disrupting its formation causes infertility and miscarriage in humans and mice, as well as chromosomal disorders like Down's syndrome. Scientists have known about this zipper structure and its importance since 1956, yet limited information is available about its shape and how it works.
Syrjänen et al. used X-ray crystallography to take images of the part of the zipper structure that interacts with the chromosomes. These images, combined with the results of biochemical and biophysical experiments, show that rod-like structures on the zipper link together sites within each chromosome. This not only allows the paired chromosomes to be held together by the zipper, but also compacts them so it's easier for them to cross over and swap genetic information.
PMCID: PMC4102245  PMID: 24950965
meiosis; chromosome structure; homologous recombination; synaptonemal complex; self-assembly; E. coli; human
2.  Tcf15 Primes Pluripotent Cells for Differentiation 
Cell Reports  2013;3(2):472-484.
The events that prime pluripotent cells for differentiation are not well understood. Inhibitor of DNA binding/differentiation (Id) proteins, which are inhibitors of basic helix-loop-helix (bHLH) transcription factor activity, contribute to pluripotency by blocking sequential transitions toward differentiation. Using yeast-two-hybrid screens, we have identified Id-regulated transcription factors that are expressed in embryonic stem cells (ESCs). One of these, Tcf15, is also expressed in the embryonic day 4.5 embryo and is specifically associated with a novel subpopulation of primed ESCs. An Id-resistant form of Tcf15 rapidly downregulates Nanog and accelerates somatic lineage commitment. We propose that because Tcf15 can be held in an inactive state through Id activity, it may prime pluripotent cells for entry to somatic lineages upon downregulation of Id. We also find that Tcf15 expression is dependent on fibroblast growth factor (FGF) signaling, providing an explanation for how FGF can prime for differentiation without driving cells out of the pluripotent state.
Graphical Abstract
► Tcf15 marks a subpopulation of pluripotent cells primed for somatic lineages ► Tcf15 expression is regulated by FGF signaling ► Tcf15 activity is repressed by Id proteins ► Tcf15 represses Nanog and drives differentiation once released from Id inhibition
Little is known about the transcription factors that oppose the pluripotency network to drive cells toward differentiation. Lowell and colleagues show that the basic helix-loop-helix (bHLH) transcription factor Tcf15 is expressed in the late preimplantation embryo and in a differentiation-primed subpopulation of embryonic stem cells and that an activated form of Tcf15 drives embryonic stem cells into a differentiation-primed state. These findings suggest a role for Tcf15 in manipulating and monitoring the earliest transition from pluripotency toward lineage commitment.
PMCID: PMC3607254  PMID: 23395635
3.  Coaching Older Adults and Carers to have their preferences Heard (COACH): A randomised controlled trial in an intermediate care setting (study protocol) 
The Australasian Medical Journal  2012;5(8):444-454.
Frail older people who are considering movement into residential aged care or returning home following a hospital admission often face complex and difficult decisions.Despite research interest in this area, a recent Cochrane review was unable to identify any studies of interventions to support decision-making in this group that met the experimental or quasi-experimental study design criteria.
This study tests the impact of a multi-component coaching intervention on the quality of preparation for care transitions, targeted to older adults and informal carers. In addition, the study assesses the impact of investing specialist geriatric resources into consultations with families in an intermediate care setting where decisions about future care needs are being made.
This study was a randomised controlled trial of 230 older adults admitted to intermediate care in Australia. Masked assessment at 3 and 12 months examined physical functioning, health–related quality of life and utilisation of health and aged care resources. A geriatrician and specialist nurse delivered a coaching intervention to both the older person and their carer/family. Components of the intervention included provision of a Question Prompt List prior to meeting with a geriatrician (to clarify medical conditions and treatments, medications, ‘red flags’, end of life decisions and options for future health care) and a follow-up meeting with a nurse who remained in telephone contact. Participants received a printed summary and an audio recording of the meeting with the geriatrician.
The costs and outcomes of the intervention are compared with usual care. Trial registration: Australian New Zealand Clinical Trials Registry (ACTRN12607000638437).
PMCID: PMC3442189  PMID: 23024719
Caregivers; continuity of patient care; cost-benefit analysis; health care costs; intermediate care facilities; patient-centred care; quality of life; randomised controlled trial
4.  Structural analysis of the human SYCE2–TEX12 complex provides molecular insights into synaptonemal complex assembly 
Open Biology  2012;2(7):120099.
The successful completion of meiosis is essential for all sexually reproducing organisms. The synaptonemal complex (SC) is a large proteinaceous structure that holds together homologous chromosomes during meiosis, providing the structural framework for meiotic recombination and crossover formation. Errors in SC formation are associated with infertility, recurrent miscarriage and aneuploidy. The current lack of molecular information about the dynamic process of SC assembly severely restricts our understanding of its function in meiosis. Here, we provide the first biochemical and structural analysis of an SC protein component and propose a structural basis for its function in SC assembly. We show that human SC proteins SYCE2 and TEX12 form a highly stable, constitutive complex, and define the regions responsible for their homotypic and heterotypic interactions. Biophysical analysis reveals that the SYCE2–TEX12 complex is an equimolar hetero-octamer, formed from the association of an SYCE2 tetramer and two TEX12 dimers. Electron microscopy shows that biochemically reconstituted SYCE2–TEX12 complexes assemble spontaneously into filamentous structures that resemble the known physical features of the SC central element (CE). Our findings can be combined with existing biological data in a model of chromosome synapsis driven by growth of SYCE2–TEX12 higher-order structures within the CE of the SC.
PMCID: PMC3411106  PMID: 22870393
synaptonemal complex; meiosis; homologous recombination; central element; SYCE2; TEX12
5.  Defining the Molecular Basis of BubR1 Kinetochore Interactions and APC/C-CDC20 Inhibition* 
The Journal of Biological Chemistry  2010;285(19):14764-14776.
BubR1 is essential for the mitotic checkpoint that prevents aneuploidy in cellular progeny by triggering anaphase delay in response to kinetochores incorrectly/not attached to the mitotic spindle. Here, we define the molecular architecture of the functionally significant N-terminal region of human BubR1 and present the 1.8 Å crystal structure of its tetratricopeptide repeat (TPR) domain. The structure reveals divergence from the classical TPR fold and is highly similar to the TPR domain of budding yeast Bub1. Shared distinctive features include a disordered loop insertion, a 310-helix, a tight turn involving glycine positive Φ angles, and noncanonical packing of and between the TPR motifs. We also define the molecular determinants of the interaction between BubR1 and kinetochore protein Blinkin. We identify a shallow groove on the concave surface of the BubR1 TPR domain that forms multiple discrete and potentially cooperative interactions with Blinkin. Finally, we present evidence for a direct interaction between BubR1 and Bub1 mediated by regions C-terminal to their TPR domains. This interaction provides a mechanism for Bub1-dependent kinetochore recruitment of BubR1. We thus present novel molecular insights into the structure of BubR1 and its interactions at the kinetochore-microtubule interface. Our studies pave the way for future structure-directed engineering aimed at dissecting the roles of kinetochore-bound and other pools of BubR1 in vivo.
PMCID: PMC2863239  PMID: 20220147
Cancer; Cell/Checkpoint; Cell/Cycle; Organisms/Human; Proteases/Ubiquitination; Protein/Protein-Protein Interactions; Protein/Protein-Protein Interactions; Protein/Structure
6.  Crystal structure of human XLF/Cernunnos reveals unexpected differences from XRCC4 with implications for NHEJ 
The EMBO Journal  2007;27(1):290-300.
The recently characterised 299-residue human XLF/Cernunnos protein plays a crucial role in DNA repair by non-homologous end joining (NHEJ) and interacts with the XRCC4–DNA Ligase IV complex. Here, we report the crystal structure of the XLF (1–233) homodimer at 2.3 Å resolution, confirming the predicted structural similarity to XRCC4. The XLF coiled-coil, however, is shorter than that of XRCC4 and undergoes an unexpected reverse in direction giving rise to a short distorted four helical bundle and a C-terminal helical structure wedged between the coiled-coil and head domain. The existence of a dimer as the major species is confirmed by size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering and other biophysical methods. We show that the XLF structure is not easily compatible with a proposed XRCC4:XLF heterodimer. However, we demonstrate interactions between dimers of XLF and XRCC4 by surface plasmon resonance and analyse these in terms of surface properties, amino-acid conservation and mutations in immunodeficient patients. Our data are most consistent with head-to-head interactions in a 2:2:1 XRCC4:XLF:Ligase IV complex.
PMCID: PMC2104711  PMID: 18046455
coiled-coil; homodimer; non-homologous end-joining (NHEJ); structure; XRCC4
7.  Interaction with the BRCA2 C-terminus Protects RAD51–DNA Filaments from Disassembly by BRC Repeats 
BRCA2 is essential for DNA repair by homologous recombination via its interaction with RAD51, mediated by short motifs in the middle and at the C-terminus of its sequence. Here we report that a conserved 36-residue exon 27 sequence of human BRCA2 (BRCA2Exon 27) interacts with RAD51 through the specific recognition of oligomerised RAD51 ATPase domains. BRCA2Exon 27 binding stabilizes the RAD51 nucleoprotein filament against disassembly by BRC repeat 4. The protection is specific for RAD51 filaments formed on single-stranded DNA and is lost when BRCA2Exon 27 is phosphorylated on Ser3291. We propose that productive recombination results from the functional balance between the different modes of interaction with RAD51 of the BRC repeat and exon 27 regions of BRCA2. Our results further suggest a mechanism whereby CDK phosphorylation of BRCA2Exon 27 at the G2-M transition alters the balance in favor of RAD51 filament disassembly, thus terminating recombination.
PMCID: PMC2096194  PMID: 17515903
BRCA2; DNA repair; Homologous Recombination; Phosphorylation; RAD51
8.  Structural biology and bioinformatics in drug design: opportunities and challenges for target identification and lead discovery 
Impressive progress in genome sequencing, protein expression and high-throughput crystallography and NMR has radically transformed the opportunities to use protein three-dimensional structures to accelerate drug discovery, but the quantity and complexity of the data have ensured a central place for informatics. Structural biology and bioinformatics have assisted in lead optimization and target identification where they have well established roles; they can now contribute to lead discovery, exploiting high-throughput methods of structure determination that provide powerful approaches to screening of fragment binding.
PMCID: PMC1609333  PMID: 16524830
structural biology; structural bioinformatics; structure-based drug design; high-throughput crystallography; virtual screening; multiprotein complexes
9.  A region of human BRCA2 containing multiple BRC repeats promotes RAD51-mediated strand exchange 
Nucleic Acids Research  2006;34(14):4000-4011.
Human BRCA2, a breast and ovarian cancer suppressor, binds to the DNA recombinase RAD51 through eight conserved BRC repeats, motifs of ∼30 residues, dispersed across a large region of the protein. BRCA2 is essential for homologous recombination in vivo, but isolated BRC repeat peptides can prevent the assembly of RAD51 into active nucleoprotein filaments in vitro, suggesting a model in which BRCA2 sequesters RAD51 in undamaged cells, and promotes recombinase function after DNA damage. How BRCA2 might fulfill these dual functions is unclear. We have purified a fragment of human BRCA2 (BRCA2BRC1–8) with 1127 residues spanning all 8 BRC repeats but excluding the C-terminal DNA-binding domain (BRCA2CTD). BRCA2BRC1–8 binds RAD51 nucleoprotein filaments in a ternary complex, indicating it may organize RAD51 on DNA. Human RAD51 is relatively ineffective in vitro at strand exchange between homologous DNA molecules unless non-physiological ions like NH4+ are present. In an ionic milieu more typical of the mammalian nucleus, BRCA2BRCI–8 stimulates RAD51-mediated strand exchange, suggesting it may be an essential co-factor in vivo. Thus, the human BRC repeats, embedded within their surronding sequences as an eight-repeat unit, mediate homologous recombination independent of the BRCA2CTD through a previously unrecognized role in control of RAD51 activity.
PMCID: PMC1557805  PMID: 16914443

Results 1-9 (9)