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Mutations in SPG7 are increasingly identified as a common cause of spastic ataxia. We describe a cohort of Irish patients with recessive SPG7-associated phenotype. Comprehensive phenotyping was performed with documentation of clinical, neurophysiological, optical coherence tomography (OCT) and genetic data from individuals with SPG7 attending two academic neurology units in Dublin, including the National Ataxia Clinic. Thirty-two symptomatic individuals from 25 families were identified. Mean age at onset was 39.1 years (range 12–61), mean disease duration 17.8 years (range 5–45), mean disease severity as quantified with the scale for the assessment and rating of ataxia 9/40 (range 3–29). All individuals displayed variable ataxia and spasticity within a spastic-ataxic phenotype, and additional ocular abnormalities. Two had spasmodic dysphonia and three had colour vision deficiency. Brain imaging consistently revealed cerebellar atrophy (n = 29); neurophysiology demonstrated a length-dependent large-fibre axonal neuropathy in 2/27 studied. The commonest variant was c.1529C > T (p.Ala510Val), present in 21 families. Five novel variants were identified. No significant thinning of average retinal nerve fibre layer (RNFL) was demonstrated on OCT (p = 0.61), but temporal quadrant reduction was evident compared to controls (p

DNA replication forks that are stalled by DNA damage activate an S-phase checkpoint that prevents irreversible fork arrest and cell death. The increased cell death caused by DNA damage in budding yeast cells lacking the Rad53 checkpoint protein kinase is partially suppressed by deletion of the EXO1 gene. Using a whole-genome sequencing approach, we identified two additional genes, RXT2 and RPH1, whose mutation can also partially suppress this DNA damage sensitivity. We provide evidence that RXT2 and RPH1 act in a common pathway, which is distinct from the EXO1 pathway. Analysis of additional mutants indicates that suppression works through the loss of the Rpd3L histone deacetylase complex. Our results suggest that the loss or absence of histone acetylation, perhaps at stalled forks, may contribute to cell death in the absence of a functional checkpoint., This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001066), the UK Medical Research Council (FC001066), and the Wellcome Trust (FC001066). This work was also funded by a European Molecular Biology Organization Long-Term Fellowship (ALTF 263–2011) to B.G.-G., and a Wellcome Trust Senior Investigator Award (106252/Z/14/Z) and a European Research Council Advanced grant (669424-CHROMOREP) to J.F.X.D.

Mutations in SPG7 are increasingly identified as a common cause of spastic ataxia. We describe a cohort of Irish patients with recessive SPG7-associated phenotype.Comprehensive phenotyping was performed with documentation of clinical, neurophysiological, optical coherence tomography (OCT) and genetic data from individuals with SPG7 attending two academic neurology units in Dublin, including the National Ataxia Clinic.Thirty-two symptomatic individuals from 25 families were identified. Mean age at onset was 39.1 years (range 12-61), mean disease duration 17.8 years (range 5-45), mean disease severity as quantified with the scale for the assessment and rating of ataxia 9/40 (range 3-29). All individuals displayed variable ataxia and spasticity within a spastic-ataxic phenotype, and additional ocular abnormalities. Two had spasmodic dysphonia and three had colour vision deficiency. Brain imaging consistently revealed cerebellar atrophy (n = 29); neurophysiology demonstrated a length-dependent large-fibre axonal neuropathy in 2/27 studied. The commonest variant was c.1529C T (p.Ala510Val), present in 21 families. Five novel variants were identified. No significant thinning of average retinal nerve fibre layer (RNFL) was demonstrated on OCT (p = 0.61), but temporal quadrant reduction was evident compared to controls (p 0.05), with significant average and temporal RNFL decline over time. Disease duration, severity and visual acuity were not correlated with RNFL thickness.Our results highlight that recessive SPG7 mutations may account for spastic ataxia with peripheral neuropathy in only a small proportion of patients. RNFL abnormalities with predominant temporal RNFL reduction are common and OCT should be considered part of the routine assessment in spastic ataxia.

Summary The eukaryotic replisome is a molecular machine that coordinates the Cdc45-MCM-GINS (CMG) replicative DNA helicase with DNA polymerases α, δ, and ε and other proteins to copy the leading- and lagging-strand templates at rates between 1 and 2 kb min−1. We have now reconstituted this sophisticated machine with purified proteins, beginning with regulated CMG assembly and activation. We show that replisome-associated factors Mrc1 and Csm3/Tof1 are crucial for in vivo rates of replisome progression. Additionally, maximal rates only occur when DNA polymerase ε catalyzes leading-strand synthesis together with its processivity factor PCNA. DNA polymerase δ can support leading-strand synthesis, but at slower rates. DNA polymerase δ is required for lagging-strand synthesis, but surprisingly also plays a role in establishing leading-strand synthesis, before DNA polymerase ε engagement. We propose that switching between these DNA polymerases also contributes to leading-strand synthesis under conditions of replicative stress., Graphical Abstract, Highlights • Reconstitution of a eukaryotic replisome capable of in vivo replication rates • Mrc1 directly stimulates replisome rate and is aided by Csm3/Tof1 • Maximum rates require leading-strand synthesis by Pol ε together with PCNA • Pol δ plays a role in the establishment of leading-strand synthesis, By reconstituting a eukaryotic replisome with purified proteins that can synthesize both leading and lagging strands at the in vivo rate, Yeeles et al. reveal the basis for rapid and efficient DNA replication by the eukaryotic replisome. Maximum rates require Mrc1 and Csm3/Tof1, and they are also dependent on leading-strand synthesis by Pol ε in the presence of PCNA. Using this system the authors show that, in addition to functioning on the lagging strand, Pol δ can play an important role in establishing leading-strand synthesis before handing over to Pol ε.

The autosomal recessive cerebellar ataxias are a heterogeneous group of neurodegenerative disorders. Mutations in the anoctamin 10 gene (ANO10) recently have been identified as a cause of autosomal recessive spinocerebellar ataxia type 10. Comprehensive phenotypic data are provided on 3 siblings with homozygous ANO10 mutations, including detailed ocular and cognitive assessments and bladder involvement not previously described in the literature. Data also are provided on unblinded therapy with coenzyme Q10, previously reported as a possible therapy in ANO10‐related ataxia. A genetic diagnosis in this family was obtained through next‐generation sequencing techniques after over 10 years of expensive sequencing of individual genes using the traditional Sanger approach. Greater commercial availability of gene panels will improve the ability to obtain a genetic diagnosis in the uncommon “non‐Friedreich's” recessive ataxias. Clinical recognition of these recessive ataxic syndromes will also inevitably improve as the full phenotypic spectrum is identified.

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

Summary The integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo., Graphical Abstract, Highlights • Reconstitution of eukaryotic chromatin replication with purified proteins • Chromatin enforces origin-specific MCM loading • FACT is essential for chromatin replication • Nucleosomes are efficiently repositioned behind the replication fork, By reconstituting chromatin replication with purified proteins, Kurat et al. show that timely progression of the replisome through chromatin requires a complex interplay between FACT, Nhp6, chromatin remodelers, and lysine acetyltransferases. Parental nucleosomes are efficiently re-assembled in the back of the replisome and positively influence lagging-strand synthesis.

The protein tyrosine phosphatase PTP1, which mediates reversible phosphorylation on tyrosine, has been shown to play an important regulatory role during Dictyostelium development. Mutants lacking PTP1 develop more rapidly than normal, while strains that overexpress PTP1 display aberrant morphology. However, the signalling pathways involved have not been characterised. In reexamining these strains, we have found that there is an inverse correlation between levels of PTP1 activity, the extent of tyrosine phosphorylation on Dictyostelium STATa after treatment with cAMP, and the proportion of the slug population exhibiting STATa nuclear enrichment in vivo. This suggests that PTP1 acts to attenuate the tyrosine phosphorylation of STATa and downstream STATa-mediated pathways. Consistent with this, we show that when PTP1 is overexpressed, there is increased expression of a prestalk cell marker at the slug posterior, a phenocopy of STATa null slugs. In ptp1 null strains, STATa tyrosine phosphorylation and nuclear enrichment in the slug anterior is increased. There is also a change in the prestalk to prespore cell ratio. Synergy experiments suggest that this is due to a cell-autonomous defect in forming the subset of prespore cells that are located in the anterior prespore region.

T.Araki and M.Gamper contributed equally to this work Dd-STAT, the protein that in part controls Dictyostelium stalk cell differentiation, is a structural and functional homolog of metazoan signal transducers and activators of transcription (STATs). Although present during growth and throughout development, Dd-STAT's tyrosine phosphorylation and nuclear localization are developmentally and spatially regulated. Prior to late aggregation, Dd-STAT is not tyrosine phosphorylated and is not selectively localized in the nucleus. During mound formation, the time at which cell-type specific gene expression initiates, Dd-STAT becomes tyrosine phosphorylated and translocates into the nuclei of all cells. The tyrosine phosphorylation and nuclear localization of Dd-STAT are induced very rapidly by extracellular cAMP through the serpentine cAMP receptor cAR1, with Dd-STAT tyrosine phosphorylation being detectable within 10 s of stimulation. This activation is independent of the only known Gβ subunit, suggesting that it may be G-protein independent. Nuclear enrichment of Dd-STAT is selectively maintained within the sub-population of prestalk cells that form the tip, the organizing center of the slug, but is lost in most of the other cells of the slug. This spatial patterning of Dd-STAT nuclear localization is consistent with its known role as a negative regulator of stalk-cell differentiation.

We present evidence that Dictyostelium slug tip cells, the pstA cells, may arise by positional differentiation, but at a site remote from that which they will eventually occupy. When first detectable, the pstA cells form a peripheral ring surrounding the other prestalk cell subtype, the pstO cells, but subsequently move above the pstO cells to form the tip. Because pstA cell differentiation requires a 10-fold higher concentration of differention-inducing factor, the stalk cell inducer, the initial patterning seems likely to reflect the existence of a morphogenetic gradient. The subsequent redistribution of the two cell types is explicable by their different rates of chemotaxis to cyclic AMP. These results help reconcile the two apparently opposing views of pattern formation in Dictyostelium, that there is positional differentiation and that pattern formation occurs by cell sorting.

Analysis of the expression patterns of two genes encoding extracellular matrix proteins shows there to be an unexpectedly complex pattern of prestalk cell differentiation and movement during the morphogenesis of Dictyostelium. The organism employs both cell sorting and positional differentiation to generate a patterned structure but these two mechanisms are used at different times during development. During slug formation prestalk cells arise at scattered positions within the aggregate and then move to its apex to form the tip. In contrast, during culmination, stalk cell differentiation occurs in a positionally localized manner at the entrance to the stalk tube. Two interacting signalling pathways regulate the differentiation of prestalk and stalk cells. Prestalk cell differentiation is induced by DIF, a chlorinated hexaphenone, and a repression mechanism prevents DIF acting to induce premature stalk cell differentiation during slug migration. At culmination intracellular cAMP levels rise, the cAMP dependent protein kinase (PKA) is activated and the block to stalk cell differentiation is lifted. Activation of PKA is also necessary in order that prestalk cells move to the entrance of the stalk tube at culmination. Thus, in Dictyostelium, PKA plays a role both in the regulation of cellular differentiation and in morphogenetic cell movement.

Replication origins in eukaryotic cells never fire more than once in a given S phase. Here, we summarize the role of cyclin–dependent kinases in limiting DNA replication origin usage to once per cell cycle in the budding yeastSaccharomyces cerevisiae. We have examined the role of different cyclins in the phosphorylation and regulation of several replication/regulatory factors including Cdc6, Sic1, ORC and DNA polymerase α–primase. In addition to being regulated by the cell cycle machinery, replication origins are also regulated by the genome integrity checkpoint kinases, Mec1 and Rad53. In response to DNA damage or drugs which interfere with the progression of replication forks, the activation of late–firing replication origins is inhibited. There is evidence indicating that the temporal programme of origin firing depends upon the local histone acetylation state. We have attempted to test the possibility that checkpoint regulation of late–origin firing operates through the regulation of the acetylation state. We found that overexpression of the essential histone acetylase, Esa1, cannot override checkpoint regulation of origin firing. We have also constructed a temperature–sensitiveesa1mutant. This mutant is unable to resume cell cycle progression after α–factor arrest. This can be overcome by overexpression of the G1 cyclin, Cln2, revealing a novel role for Esa1 in regulating Start.

Prestalk cell differentiation in Dictyostelium is induced by DIF and two DIF-induced genes, ecmA and ecmB, have revealed the existence of multiple prestalk and stalk cell sub-types. These different sub-types are defined by the pattern of expression of subfragments derived from the ecmA and ecmB promoters. These markers have been utilised in three ways; for fate mapping in vivo, to investigate the molecular mechanisms underlying DIF signalling and to explore the relative requirement for DIF and other signalling molecules for prestalk and stalk cell differentiation in vitro. The heterogeneity of the prestalk and stalk populations seems to be reflected in differences in the cell signalling pathways that they utilise.

In theDictyosteliumslug there are two types of prestalk cells, pstA cells and pstO cells, that differ in their ability to utilize the distal and proximal parts of the promoter ofecmA,a gene that is specifically expressed in prestalk cells. When Rm, a dominant inhibitory form of the regulatory subunit of cAMP-dependent protein kinase (PKA), is expressed under the control of the complete promoter of theecmAgene (in a construct termed ecmAO:Rm) development proceeds to the slug stage. Although able to form small but outwardly normal slugs, ecmAO:Rm cells are defective in prestalk cell differentiation. In ecmAO:Rm cells, the induction of pstA- and pstO-specific gene expression by the stalk cell inducer DIF is greatly inhibited. Paradoxically, a very large fraction of the cells in an ecmAO:Rm slug show evidence of once having expressed the ecmA and ecmO prestalk markers. However, we present evidence that this is due to abortive prestalk cell differentiation that terminates when sufficient Rm protein has accumulated to block PKA activity. This results in regulative transdifferentiation of prespore cells to form prestalk cells. During their transitory period as prestalk cells the ecmAO:Rm cells coexpress both the ecmA and ecmO markers, indicating a possible link between PKA activity and divergence of the two prestalk cell subtypes. Finally, we show that the level of the DNA binding activity believed to lie at the end of the DIF signal transduction pathway is reduced in ecmAO:Rm slugs.

The ecmA gene is expressed in Dictyostelium prestalk cells and is inducible by differentiation-inducing factor (DIF), a low-molecular-weight lipophilic substance. The ecmB gene is expressed in stalk cells and is under negative control by two repressor elements. Each repressor element contains two copies of the sequence TTGA in an inverted relative orientation. There are activator elements in the ecmA promoter that also contain two TTGA sequences, but in the same relative orientation. Gel retardation assays suggest that the same protein binds to the ecmB repressor and the ecmA activator. We propose that DIF induces prestalk cell differentiation by activating this protein and that the protein also binds to the promoters of stalk-specific genes, acting as a repressor that holds cells in the prestalk state until culmination is triggered.

T-lymphocyte development takes place mainly in the thymus, where stromal cells of epithelial and haemopoietic origin are involved in inductive and selective mechanisms, which enable specific lymphocyte populations to migrate to the periphery and establish a network of immune responses. Experiments with intact animals have clarified the precursor-product relationships between thymocyte subpopulations, but the molecular mechanisms of cell interactions in the thymus are difficult to study in vivo. In an attempt to expand thymic cell populations in vivo and maintain them in vitro for such studies, we directed high levels of expression of the murine c-myc proto-oncogene in transgenic mice by inserting it into the mouse Thy-1 transcriptional unit. Such mice develop thymic tumours which contain proliferating thymocytes and, interestingly, expanded populations of epithelial cells. Both cell types can be maintained in vitro.