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1.
Tomography ; 7(4): 915-931, 2021 12 08.
Article in English | MEDLINE | ID: mdl-34941648

ABSTRACT

Ocular abnormalities occur frequently in Friedreich's ataxia (FRDA), although visual symptoms are not always reported. We evaluated a cohort of patients with FRDA to characterise the clinical phenotype and optic nerve findings as detected with optical coherence tomography (OCT). A total of 48 patients from 42 unrelated families were recruited. Mean age at onset was 13.8 years (range 4-40), mean disease duration 19.5 years (range 5-43), mean disease severity as quantified with the Scale for the Assessment and Rating of Ataxia 22/40 (range 4.5-38). All patients displayed variable ataxia and two-thirds had ocular abnormalities. Statistically significant thinning of average retinal nerve fibre layer (RNFL) and thinning in all but the temporal quadrant compared to controls was demonstrated on OCT. Significant RNFL and macular thinning was documented over time in 20 individuals. Disease severity and visual acuity were correlated with RNFL and macular thickness, but no association was found with disease duration. Our results highlight that FDRA is associated with subclinical optic neuropathy. This is the largest longitudinal study of OCT findings in FRDA to date, demonstrating progressive RNFL thickness decline, suggesting that RNFL thickness as measured by OCT has the potential to become a quantifiable biomarker for the evaluation of disease progression in FRDA.


Subject(s)
Friedreich Ataxia , Optic Nerve Diseases , Friedreich Ataxia/complications , Friedreich Ataxia/diagnostic imaging , Humans , Longitudinal Studies , Optic Nerve Diseases/complications , Optic Nerve Diseases/diagnosis , Tomography, Optical Coherence/methods , Visual Acuity
3.
J Neurol ; 268(10): 3897-3907, 2021 Oct.
Article in English | MEDLINE | ID: mdl-33774748

ABSTRACT

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.


Subject(s)
Muscle Spasticity , Spastic Paraplegia, Hereditary , ATPases Associated with Diverse Cellular Activities/genetics , Adolescent , Adult , Child , Child, Preschool , Humans , Intellectual Disability , Metalloendopeptidases/genetics , Middle Aged , Muscle Spasticity/diagnostic imaging , Muscle Spasticity/genetics , Neurophysiology , Optic Atrophy , Phenotype , Spastic Paraplegia, Hereditary/diagnostic imaging , Spastic Paraplegia, Hereditary/genetics , Spinocerebellar Ataxias , Tomography, Optical Coherence , Young Adult
4.
Genetics ; 211(2): 503-513, 2019 02.
Article in English | MEDLINE | ID: mdl-30559326

ABSTRACT

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.


Subject(s)
DNA Damage , Histone Deacetylase 1/genetics , Histone Deacetylases/genetics , Histone Demethylases/genetics , Repressor Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , DNA Replication , Histone Deacetylase 1/metabolism , Histone Demethylases/metabolism , Mutation , Repressor Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism
5.
Nat Commun ; 9(1): 5061, 2018 11 29.
Article in English | MEDLINE | ID: mdl-30498216

ABSTRACT

Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase. A key step is the recruitment of GINS that requires the leading-strand polymerase Pol epsilon, composed of Pol2, Dpb2, Dpb3, Dpb4. While a truncation of the catalytic N-terminal Pol2 supports cell division, Dpb2 and C-terminal Pol2 (C-Pol2) are essential for viability. Dpb2 and C-Pol2 are non-catalytic modules, shown or predicted to be related to an exonuclease and DNA polymerase, respectively. Here, we present the cryo-EM structure of the isolated C-Pol2/Dpb2 heterodimer, revealing that C-Pol2 contains a DNA polymerase fold. We also present the structure of CMG/C-Pol2/Dpb2 on a DNA fork, and find that polymerase binding changes both the helicase structure and fork-junction engagement. Inter-subunit contacts that keep the helicase-polymerase complex together explain several cellular phenotypes. At least some of these contacts are preserved during Pol epsilon-dependent CMG assembly on path to origin firing, as observed with DNA replication reconstituted in vitro.


Subject(s)
DNA Polymerase II/chemistry , DNA Polymerase II/metabolism , DNA Replication/physiology , DNA-Binding Proteins/metabolism , DNA/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA/chemistry , DNA/genetics , DNA Polymerase II/genetics , DNA Replication/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Humans , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Structure, Tertiary
6.
Mov Disord Clin Pract ; 4(2): 258-262, 2017.
Article in English | MEDLINE | ID: mdl-30838263

ABSTRACT

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.

7.
Mol Cell ; 65(1): 117-130, 2017 Jan 05.
Article in English | MEDLINE | ID: mdl-27989438

ABSTRACT

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.


Subject(s)
Chromatin/genetics , DNA Replication , DNA, Fungal/genetics , Nucleosomes/genetics , Replication Origin , Saccharomyces cerevisiae/genetics , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Chromatin/metabolism , DNA Polymerase I/genetics , DNA Polymerase I/metabolism , DNA, Fungal/biosynthesis , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , HMGN Proteins/genetics , HMGN Proteins/metabolism , High Mobility Group Proteins/genetics , High Mobility Group Proteins/metabolism , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Minichromosome Maintenance Proteins/genetics , Minichromosome Maintenance Proteins/metabolism , Nucleosomes/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Time Factors , Transcriptional Elongation Factors/genetics , Transcriptional Elongation Factors/metabolism
8.
Mol Cell ; 65(1): 105-116, 2017 Jan 05.
Article in English | MEDLINE | ID: mdl-27989442

ABSTRACT

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.


Subject(s)
DNA Replication , DNA, Fungal/genetics , Saccharomyces cerevisiae/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA Polymerase II/genetics , DNA Polymerase II/metabolism , DNA Polymerase III/genetics , DNA Polymerase III/metabolism , DNA, Fungal/biosynthesis , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Time Factors
9.
Nature ; 519(7544): 431-5, 2015 Mar 26.
Article in English | MEDLINE | ID: mdl-25739503

ABSTRACT

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.


Subject(s)
DNA Replication , Replication Origin/physiology , Saccharomyces cerevisiae Proteins/isolation & purification , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Cell Cycle Proteins/metabolism , Cyclin-Dependent Kinases/metabolism , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Minichromosome Maintenance Proteins/metabolism , Multienzyme Complexes/metabolism , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Nuclear Proteins/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Replication Origin/genetics , Replication Protein A/metabolism , Saccharomyces cerevisiae/enzymology
10.
Philos Trans R Soc Lond B Biol Sci ; 359(1441): 31-8, 2004 Jan 29.
Article in English | MEDLINE | ID: mdl-15065654

ABSTRACT

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 yeast Saccharomyces 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 alpha-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, Esal, cannot override checkpoint regulation of origin firing. We have also constructed a temperature-sensitive esa1 mutant. This mutant is unable to resume cell cycle progression after alpha-factor arrest. This can be overcome by overexpression of the G1 cyclin, Cln2, revealing a novel role for Esal in regulating Start.


Subject(s)
Cell Cycle Proteins , Cell Cycle/physiology , Cyclin-Dependent Kinases/metabolism , DNA Damage/physiology , DNA Replication , Replication Origin/physiology , Acetyltransferases/metabolism , Checkpoint Kinase 2 , Histone Acetyltransferases , Histones/metabolism , Intracellular Signaling Peptides and Proteins , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/metabolism
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