Gregory A. Farnum

Clustering of Alpers disease mutations and catalytic defects in biochemical variants reveal new features of molecular mechanism of the human mitochondrial replicase, Pol γ

Mutations in Pol γ represent a major cause of human mitochondrial diseases, especially those affecting the nervous system in adults and in children. Recessive mutations in Pol γ represent nearly half of those reported to date, and they are nearly uniformly distributed along the length of the POLG1 gene (Human DNA Polymerase gamma Mutation Database); the majority of them are linked to the most severe form of POLG syndrome, Alpers–Huttenlocher syndrome. In this report, we assess the structure–function relationships for recessive disease mutations by reviewing existing biochemical data on site-directed mutagenesis of the human, Drosophila and yeast Pol γs, and their homologs from the family A DNA polymerase group. We do so in the context of a molecular model of Pol γ in complex with primer–template DNA, which we have developed based upon the recently solved crystal structure of the apoenzyme form. We present evidence that recessive mutations cluster within five distinct functional modules in the catalytic core of Pol γ. Our results suggest that cluster prediction can be used as a diagnosis-supporting tool to evaluate the pathogenic role of new Pol γ variants.

Mapping 136 pathogenic mutations into functional modules in human DNA polymerase γ establishes predictive genotype-phenotype correlations for the complete spectrum of POLG syndromes.

We establish the genotype-phenotype correlations for the complete spectrum of POLG syndromes by refining our previously described protocol for mapping pathogenic mutations in the human POLG gene to functional clusters in the catalytic core of the mitochondrial replicase, Pol γ (1). We assigned 136 mutations to five clusters and identify segments of primary sequence that can be used to delimit the boundaries of each cluster. We report that compound heterozygotes with two mutations from different clusters manifested more severe, earlier-onset POLG syndromes, whereas two mutations from the same cluster are less common and generally are associated with less severe, later onset POLG syndromes. We also show that specific cluster combinations are more severe than others and have a higher likelihood to manifest at an earlier age. Our clustering method provides a powerful tool to predict the pathogenic potential and predicted disease phenotype of novel variants and mutations in POLG, the most common nuclear gene underlying mitochondrial disorders. We propose that such a prediction tool would be useful for routine diagnostics for mitochondrial disorders. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Structure and physical properties of substituted malonate divalent metal coordination polymers with dipyridylamine co-ligands: acentric chain, herringbone layer, and novel binodal network topologies

Three divalent metal coordination polymers and one molecular species containing 4,4′-dipyridylamine (dpa) and substituted malonate ligands have been prepared and structurally characterized by single crystal X-ray diffraction. The degree of substitution and different coordination environments promote significant structural diversity in this system. {[Cu(mmal)(Hmmal)(Hdpa)]·H2O}n (1, mmal = methylmalonate) crystallizes in an acentric space group and possesses 1-D coordination polymer chain motifs containing [Cu(OCO)]n linkages. {[Cu3(dmmal)2(dpa)3](ClO4)2·2H2O}n (2, dmmal = dimethylmalonate) manifests an unprecedented (4,5)-connected binodal network with (4462)(4664)2 topology, and contains equatorial–equatorial bridged linear {Cu3O2} trimeric units. {[Cd2(dmmal)2(dpa)(Hdpa)](ClO4)·2H2O·CH3CH2OH}n (3) displays (6,3) herringbone layers, while {[Co(dmmal)2(Hdpa)2]·6H2O} (4) has co-crystallized neutral coordination complexes and hydrogen-bonded hexameric water molecule aggregations. Ultraviolet irradiation of a sample of 3 resulted in blue-violet light emission due to π–π* transitions within the dpa and Hdpa+ ligands. Variable temperature magnetic studies indicated antiferromagnetic coupling along the {Cu(OCO)}n chains in 1 and within the {Cu3O2} linear trimers in 2.

The N-terminal Domain of the Drosophila Mitochondrial Replicative DNA Helicase Contains an Iron-Sulfur Cluster and Binds DNA

Background: Despite high evolutionary conservation, the function of the N-terminal domain (NTD) of mtDNA helicase remains elusive. Results: Drosophila NTD contains an iron-sulfur cluster and binds DNA. Conclusion: The iron-sulfur cluster in mtDNA helicase enhances protein stability, and may regulate its biological functions. Significance: Discovery of an Fe-S cluster in insect mtDNA helicase presents a novel opportunity to explore species-specific relationships in the replisome. The metazoan mitochondrial DNA helicase is an integral part of the minimal mitochondrial replisome. It exhibits strong sequence homology with the bacteriophage T7 gene 4 protein primase-helicase (T7 gp4). Both proteins contain distinct N- and C-terminal domains separated by a flexible linker. The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain. Whereas the N-terminal domain in T7 gp4 contains a DNA primase activity, this function is lost in metazoan mtDNA helicase. Thus, although the functions of the C-terminal domain and the linker are partially understood, the role of the N-terminal region in the metazoan replicative mtDNA helicase remains elusive. Here, we show that the N-terminal domain of Drosophila melanogaster mtDNA helicase coordinates iron in a 2Fe-2S cluster that enhances protein stability in vitro. The N-terminal domain binds the cluster through conserved cysteine residues (Cys68, Cys71, Cys102, and Cys105) that are responsible for coordinating zinc in T7 gp4. Moreover, we show that the N-terminal domain binds both single- and double-stranded DNA oligomers, with an apparent Kd of ∼120 nm. These findings suggest a possible role for the N-terminal domain of metazoan mtDNA helicase in recruiting and binding DNA at the replication fork.

Pathogenicity in POLG syndromes: DNA polymerase gamma pathogenicity prediction server and database

DNA polymerase gamma (POLG) is the replicative polymerase responsible for maintaining mitochondrial DNA (mtDNA). Disorders related to its functionality are a major cause of mitochondrial disease. The clinical spectrum of POLG syndromes includes Alpers-Huttenlocher syndrome (AHS), childhood myocerebrohepatopathy spectrum (MCHS), myoclonic epilepsy myopathy sensory ataxia (MEMSA), the ataxia neuropathy spectrum (ANS) and progressive external ophthalmoplegia (PEO). We have collected all publicly available POLG-related patient data and analyzed it using our pathogenic clustering model to provide a new research and clinical tool in the form of an online server. The server evaluates the pathogenicity of both previously reported and novel mutations. There are currently 176 unique point mutations reported and found in mitochondrial patients in the gene encoding the catalytic subunit of POLG, POLG. The mutations are distributed nearly uniformly along the length of the primary amino acid sequence of the gene. Our analysis shows that most of the mutations are recessive, and that the reported dominant mutations cluster within the polymerase active site in the tertiary structure of the POLG enzyme. The POLG Pathogenicity Prediction Server (http://polg.bmb.msu.edu) is targeted at clinicians and scientists studying POLG disorders, and aims to provide the most current available information regarding the pathogenicity of POLG mutations.

Poly[4,4′-imino­dipyridinium [tetra-μ3-oxido-tetra­oxido-di-μ4-phosphato-κ4O:O′:O′′:O′′′-tetra­vanadium(V)]]

In the title salt, {(C10H11N3)[V4O8(PO4)2]}n, cubane-like [V4O8]4+ clusters are connected by phosphate anions into anionic [V4P2O16]n 2n− layers. These aggregate into the three-dimensional structure via N—H⋯O hydrogen-bonding mechanisms imparted by 4,4′-iminodipyridinium dications situated between the layers.

Bis(dimethyl­malonato-κ2O,O′)bis­[4-(4-pyridylamino-κN4)pyridinium]nickel(II) hexa­hydrate

In the title compound, [Ni(C5H6O4)2(C10H10N3)2]·6H2O, divalent nickel ions situated on the crystallographic twofold axis are octahedrally coordinated by four O atoms from two dimethylmalonate ligands in a 1,3-chelating mode and two N atoms from two protonated monodentate 4,4′-dipyridylamine molecules. The molecules link into chains via N—H⋯O hydrogen bonding mediated by protonated pyridyl groups. The chains form layer patterns via π–π stacking [centroid–centroid distance = 3.777 (2) Å] . Water molecule hexamers are generated from the unligated water molecules (three per asymmetric unit) by inversion centers at Wyckoff position d. These clusters are situated between the pseudolayers, providing hydrogen-bonding pathways that build up the three-dimensional structure.

1,4-Bis(4-pyridylmeth­yl)piperazin-1-ium perchlorate fumaric acid hemisolvate

In the title salt, C16H21N4 +·ClO4 −·0.5C4H4O4, fumaric acid molecules, situated across crystallographic inversion centres, are O—H⋯N hydrogen bonded to two protonated 1,4-bis(4-pyridylmethyl)piperazine cations, forming trimolecular units. These construct one-dimensional supramolecular ribbons by N—H⋯N hydrogen bonding, and further aggregate via π–π interactions [shortest C⋯C contact = 3.640 (1) Å] and perchlorate-mediated C—H⋯O interactions.

4-(4-Pyridylamino)pyridinium perchlorate

In the title salt, C10H10N3 +·ClO4 −, the 4-(4-pyridylamino)pyridinium cations are linked into chains via N—H⋯N hydrogen bonding and into layers by C—H⋯π interactions [C⋯Cg = 3.3875 (19) Å]. Perchlorate ions are anchored to the layer motifs by N—H⋯O hydrogen bonding. The perchlorate anion was found to be disordered about a Cl—O axis, with two sites, each of equal occupancy, being resolved for the three remaining O atoms.

Bis(perchlorato-κO)tetra­kis[1-(2-pyridyl)-4-(4-pyridylmethyl-κN)piperazine]cadmium(II)

In the title compound, [Cd(ClO4)2(C15H18N4)4], the CdII ion is coordinated in a slightly distorted octahedral environment by two trans monodentate perchlorate ligands and four 1-(2-pyridyl)-4-(4-pyridylmethyl)piperazine (pmpp) ligands. In the crystal structure, molecules are organized into layers parallel to the ab plane by C—H⋯O interactions. Similar interactions promote the stacking of these layers into the three-dimensional crystal structure.