Publications

Analysis of HGD Gene Mutations in Patients with Alkaptonuria from the United Kingdom: Identification of Novel Mutations.

Publications by the Blundell group - Mon, 23/02/2015 - 18:58
Related Articles

Analysis of HGD Gene Mutations in Patients with Alkaptonuria from the United Kingdom: Identification of Novel Mutations.

JIMD Rep. 2015 Feb 15;

Authors: Usher JL, Ascher DB, Pires DE, Milan AM, Blundell TL, Ranganath LR

Abstract
Alkaptonuria (AKU) is a rare autosomal recessive disorder with incidence ranging from 1:100,000 to 1:250,000. The disorder is caused by a deficiency of the enzyme homogentisate 1,2-dioxygenase (HGD), which results from defects in the HGD gene. This enzyme converts homogentisic acid to maleylacetoacetate and has a major role in the catabolism of phenylalanine and tyrosine. To elucidate the mutation spectrum of the HGD gene in patients with alkaptonuria from 42 patients attending the National Alkaptonuria Centre, 14 exons of the HGD gene and the intron-exon boundaries were analysed by PCR-based sequencing. A total of 34 sequence variants was observed, confirming the genetic heterogeneity of AKU. Of these mutations, 26 were missense substitutions and four splice site mutations. There were two deletions and one duplication giving rise to frame shifts and one substitution abolishing the translation termination codon (no stop). Nine of the mutations were previously unreported novel variants. Using computational approaches based on the 3D structure, these novel mutations are predicted to affect the activity of the protein complex through destabilisation of the individual protomer structure or through disruption of protomer-protomer interactions.

PMID: 25681086 [PubMed - as supplied by publisher]

Categories: Publications

DNA repair. PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair.

Publications by the Blundell group - Tue, 10/02/2015 - 16:17
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DNA repair. PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair.

Science. 2015 Jan 9;347(6218):185-8

Authors: Ochi T, Blackford AN, Coates J, Jhujh S, Mehmood S, Tamura N, Travers J, Wu Q, Draviam VM, Robinson CV, Blundell TL, Jackson SP

Abstract
XRCC4 and XLF are two structurally related proteins that function in DNA double-strand break (DSB) repair. Here, we identify human PAXX (PAralog of XRCC4 and XLF, also called C9orf142) as a new XRCC4 superfamily member and show that its crystal structure resembles that of XRCC4. PAXX interacts directly with the DSB-repair protein Ku and is recruited to DNA-damage sites in cells. Using RNA interference and CRISPR-Cas9 to generate PAXX(-/-) cells, we demonstrate that PAXX functions with XRCC4 and XLF to mediate DSB repair and cell survival in response to DSB-inducing agents. Finally, we reveal that PAXX promotes Ku-dependent DNA ligation in vitro and assembly of core nonhomologous end-joining (NHEJ) factors on damaged chromatin in cells. These findings identify PAXX as a new component of the NHEJ machinery.

PMID: 25574025 [PubMed - in process]

Categories: Publications

The spatial organization of non-homologous end joining: from bridging to end joining.

Publications by the Blundell group - Tue, 13/01/2015 - 01:25
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The spatial organization of non-homologous end joining: from bridging to end joining.

DNA Repair (Amst). 2014 May;17:98-109

Authors: Ochi T, Wu Q, Blundell TL

Abstract
Non-homologous end joining (NHEJ) repairs DNA double-strand breaks generated by DNA damage and also those occurring in V(D)J recombination in immunoglobulin and T cell receptor production in the immune system. In NHEJ DNA-PKcs assembles with Ku heterodimer on the DNA ends at double-strand breaks, in order to bring the broken ends together and to assemble other proteins, including DNA ligase IV (LigIV), required for DNA repair. Here we focus on structural aspects of the interactions of LigIV with XRCC4, XLF, Artemis and DNA involved in the bridging and end-joining steps of NHEJ. We begin with a discussion of the role of XLF, which interacts with Ku and forms a hetero-filament with XRCC4; this likely forms a scaffold bridging the DNA ends. We then review the well-defined interaction of XRCC4 with LigIV, and discuss the possibility of this complex interrupting the filament formation, so positioning the ligase at the correct positions close to the broken ends. We also describe the interactions of LigIV with Artemis, the nuclease that prepares the ends for ligation and also interacts with DNA-PK. Lastly we review the likely affects of Mendelian mutations on these multiprotein assemblies and their impacts on the form of inherited disease.

PMID: 24636752 [PubMed - indexed for MEDLINE]

Categories: Publications

Structural and physiological analyses of the alkanesulphonate-binding protein (SsuA) of the citrus pathogen Xanthomonas citri.

Publications by the Blundell group - Mon, 05/01/2015 - 17:46
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Structural and physiological analyses of the alkanesulphonate-binding protein (SsuA) of the citrus pathogen Xanthomonas citri.

PLoS One. 2013;8(11):e80083

Authors: Tófoli de Araújo F, Bolanos-Garcia VM, Pereira CT, Sanches M, Oshiro EE, Ferreira RC, Chigardze DY, Barbosa JA, de Souza Ferreira LC, Benedetti CE, Blundell TL, Balan A

Abstract
BACKGROUND: The uptake of sulphur-containing compounds plays a pivotal role in the physiology of bacteria that live in aerobic soils where organosulfur compounds such as sulphonates and sulphate esters represent more than 95% of the available sulphur. Until now, no information has been available on the uptake of sulphonates by bacterial plant pathogens, particularly those of the Xanthomonas genus, which encompasses several pathogenic species. In the present study, we characterised the alkanesulphonate uptake system (Ssu) of Xanthomonas axonopodis pv. citri 306 strain (X. citri), the etiological agent of citrus canker.
METHODOLOGY/PRINCIPAL FINDINGS: A single operon-like gene cluster (ssuEDACB) that encodes both the sulphur uptake system and enzymes involved in desulphurisation was detected in the genomes of X. citri and of the closely related species. We characterised X. citri SsuA protein, a periplasmic alkanesulphonate-binding protein that, together with SsuC and SsuB, defines the alkanesulphonate uptake system. The crystal structure of SsuA bound to MOPS, MES and HEPES, which is herein described for the first time, provides evidence for the importance of a conserved dipole in sulphate group coordination, identifies specific amino acids interacting with the sulphate group and shows the presence of a rather large binding pocket that explains the rather wide range of molecules recognised by the protein. Isolation of an isogenic ssuA-knockout derivative of the X. citri 306 strain showed that disruption of alkanesulphonate uptake affects both xanthan gum production and generation of canker lesions in sweet orange leaves.
CONCLUSIONS/SIGNIFICANCE: The present study unravels unique structural and functional features of the X. citri SsuA protein and provides the first experimental evidence that an ABC uptake system affects the virulence of this phytopathogen.

PMID: 24282519 [PubMed - indexed for MEDLINE]

Categories: Publications
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