SUMMARY Active genes in yeast can be targeted to the nuclear periphery through interaction of cis-acting “DNA zip codes” with the nuclear pore complex. We find that genes with identical zip codes cluster together. This clustering was specific; pairs of genes that were targeted to the nuclear periphery by different zip codes did not cluster together. Insertion of two different zip codes (GRS I or GRS III) at an ectopic site induced clustering with endogenous genes having that zip code. Targeting to the nuclear periphery and interaction with the nuclear pore is a pre-requisite for gene clustering, but clustering can be maintained in the nucleoplasm. Finally, we find that the Put3 transcription factor recognizes the GRS I zip code to mediate both targeting to the NPC and interchromosomal clustering. These results suggest that zip code-mediated clustering of genes at the nuclear periphery influences the three-dimensional arrangement of the yeast genome.

The human mitochondrial genome is replicated by DNA polymerase γ in concert with key components of the mitochondrial DNA (mtDNA) replication machinery. Defects in mtDNA replication or nucleotide metabolism cause deletions, point mutations, or depletion of mtDNA. The resulting loss of cellular respiration ultimately induces mitochondrial genetic diseases, including mtDNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and mtDNA deletion disorders such as progressive external ophthalmoplegia, ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy. Here we review the current literature regarding human mtDNA replication and heritable disorders caused by genetic changes of the POLG, POLG2, Twinkle, RNASEH1, DNA2 and MGME1 genes.

Background: Mitochondrial porins, or voltage-dependent anion-selective channels (VDAC) allow the passage of small molecules across the mitochondrial outer membrane, and are involved in complex interactions regulating organellar and cellular metabolism. Numerous organisms possess multiple porin isoforms, and initial studies indicated an intriguing evolutionary history for these proteins and the genes that encode them.

Defects in mitochondrial DNA (mtDNA) maintenance comprise an expanding repertoire of polymorphic diseases caused, in part, by mutations in the genes encoding the p140 mtDNA polymerase (POLG), its p55 accessory subunit (POLG2) or the mtDNA helicase (C10orf2). In an exploration of nuclear genes for mtDNA maintenance linked to mitochondrial disease, eight heterozygous mutations (six novel) in POLG2 were identified in one control and eight patients with POLG-related mitochondrial disease that lacked POLG mutations. Of these eight mutations, we biochemically characterized seven variants [c.307G>A (G103S); c.457C>G (L153V); c.614C>G (P205R); c.1105A>G (R369G); c.1158T>G (D386E); c.1268C>A (S423Y); c.1423_1424delTT (L475DfsX2)] that were previously uncharacterized along with the wild-type protein and the G451E pathogenic variant. These seven mutations encode amino acid substitutions that map throughout the protein, including the p55 dimer interface and the C-terminal domain that interacts with the catalytic subunit. Recombinant proteins harboring these alterations were assessed for stimulation of processive DNA synthesis, binding to the p140 catalytic subunit, binding to dsDNA and self-dimerization. Whereas the G103S, L153V, D386E and S423Y proteins displayed wild-type behavior, the P205R and R369G p55 variants had reduced stimulation of processivity and decreased affinity for the catalytic subunit. Additionally, the L475DfsX2 variant, which possesses a C-terminal truncation, was unable to bind the p140 catalytic subunit, unable to bind dsDNA and formed aberrant oligomeric complexes. Our biochemical analysis helps explain the pathogenesis of POLG2 mutations in mitochondrial disease and emphasizes the need to quantitatively characterize the biochemical consequences of newly discovered mutations before classifying them as pathogenic.

Removal of substrate (+)-camphor from the active site of cytochrome P450cam (CYP101A1) results in nuclear magnetic resonance-detected perturbations in multiple regions of the enzyme. The 1H,15N correlation map of substrate-free diamagnetic Fe(II) CO-bound CYP101A permits these perturbations to be mapped onto the solution structure of the enzyme. Residual dipolar couplings (RDCs) were measured for 15N-1H amide pairs in two independent alignment media for the substrate-free enzyme and used as restraints in solvated molecular dynamics (MD) simulations to generate an ensemble of best-fit structures of the substrate-free enzyme in solution. NMR-detected chemical shift perturbations reflect changes in the electronic environment of the NH pairs, such as hydrogen bonding and ring current shifts, and are observed for residues in the active site as well as in hinge regions between secondary structural features. RDCs provide information regarding relative orientations of secondary structures, and RDC-restrained MD simulations indicate that portions of a β-rich region adjacent to the active site shift so as to partially occupy the vacancy left by removal of substrate. The accessible volume of the active site is reduced in the substrate-free enzyme relative to the substrate-bound structure calculated using the same methods. Both symmetric and asymmetric broadening of multiple resonances observed upon substrate removal as well as localized increased errors in RDC fits suggest that an ensemble of enzyme conformations are present in the substrate-free form.

Expeller-pressed (EP) canola meal contains more residual oil than solvent-extracted canola meal and might be an attractive feedstuff for swine, but it has been poorly characterized. In Exp. 1, six ileal-cannulated barrows (36 kg of BW) were fed at 3x maintenance either a 44% EP canola meal diet or a N-free diet in a crossover design to measure energy and AA digestibility and calculate standardized ileal digestible (SID) AA and NE content, with 6 observations per diet. Each period consisted of a 5-d diet adaptation and a 2-d feces and 3-d digesta collection. The EP canola meal contained (% of DM) 38.5% CP, 13.3% ether extract, 2.42% Lys, 1.54% Thr, 0.62% Met, and 23.2 micromol/g of glucosinolates. Apparent total tract energy digestibility was 75.0% and the DE and predicted NE content were 3.77 and 2.55 Mcal/kg (in DM), respectively. The SID AA content (% of DM) was 1.77% Lys, 1.04% Thr, and 0.52% Met. In Exp. 2, a total of 1,100 pigs (25 kg of BW) housed in 50 pens were fed 5 dietary regimens with 0, 7.5, 15, and 22.5% or decreasing amounts (22.5, 15, 7.5, and 0%, respectively) of EP canola meal over 4 phases to validate performance and carcass characteristics. Diets were formulated to contain equal NE:SID Lys for each growth phase (g/Mcal; 4.04, d 0 to 25; 3.63, d 26 to 50; 3.23, d 51 to 77; 2.83, d 78 to 90). At slaughter, carcass characteristics were measured for all pigs, and jowl fat was sampled for 2 pigs per pen. For d 51 to 90, the 22.5% EP canola meal regimen was reduced to 18% (22.5/18%) because of decreased ADFI in phases 1 and 2. Overall (d 0 to 90), increasing dietary EP canola meal linearly decreased (P < 0.001) ADG and ADFI and linearly increased (P < 0.01) G:F. For 0 and 22.5/18% EP canola meal, respectively, ADG was 978 and 931 g/d, ADFI was 2.77 and 2.58 kg/d, and G:F was 0.366 and 0.378. Increasing dietary EP canola meal did not alter the carcass backfat thickness, loin depth, or jowl fat fatty acid profile. Pigs fed 22.5/18% EP canola meal reached slaughter weight 3 d after (P < 0.05) pigs fed 0% EP canola meal. In summary, EP canola meal provided adequate energy and AA; however, ADG was reduced by 3 g/d per 1% of EP canola meal inclusion, likely because of increased dietary glucosinolates. Thus, the amount of EP canola meal included in swine diets should be targeted to an expected growth performance and carcass quality. Finally, diets formulated to contain an equal NE and SID AA content did not entirely eliminate the risks for reduced growth performance associated with inclusion of an alternative feedstuff.

Mammalian mitochondrial DNA (mtDNA) is replicated by the heterotrimeric Pol γ comprised of a single catalytic subunit, encoded by Polg, and a homodimeric accessory subunit encoded by the Polg2 gene. While the catalytic subunit has been shown to be essential for embryo development, genetic data regarding the accessory subunit are lacking in mammalian systems. Here, we describe the generation of heterozygous (Polg2(+/-)) and homozygous (Polg2(-/-)) knockout (KO) mice. Polg2(+/-) mice are haplosufficient and develop normally with no discernable difference in mitochondrial function through 2 years of age. In contrast, the Polg2(-/-) is embryonic lethal at day 8.0-8.5 p.c. with concomitant loss of mtDNA and mtDNA gene products. Electron microscopy shows severe ultra-structural defects and loss of organized cristae in mitochondria of the Polg2(-/-) embryos as well as an increase in lipid accumulation compared with both wild-type (WT) and Polg2(+/-) embryos. Our data indicate that Polg2 function is critical to mammalian embryogenesis and mtDNA replication, and that a single copy of Polg2 is sufficient to sustain life.

Nucleoside reverse transcriptase inhibitors (NRTIs) were the first drugs used to treat human immunodeficiency virus (HIV) the cause of acquired immunodeficiency syndrome. Development of severe mitochondrial toxicity has been well documented in patients infected with HIV and administered NRTIs. In vitro biochemical experiments have demonstrated that the replicative mitochondrial DNA (mtDNA) polymerase gamma, Polg, is a sensitive target for inhibition by metabolically active forms of NRTIs, nucleotide reverse transcriptase inhibitors (NtRTIs). Once incorporated into newly synthesized daughter strands NtRTIs block further DNA polymerization reactions. Human cell culture and animal studies have demonstrated that cell lines and mice exposed to NRTIs display mtDNA depletion. Further complicating NRTI off-target effects on mtDNA maintenance, two additional DNA polymerases, Pol beta and PrimPol, were recently reported to localize to mitochondria as well as the nucleus. Similar to Polg, in vitro work has demonstrated both Pol beta and PrimPol incorporate NtRTIs into nascent DNA. Cell culture and biochemical experiments have also demonstrated that antiviral ribonucleoside drugs developed to treat hepatitis C infection act as off-target substrates for POLRMT, the mitochondrial RNA polymerase and primase. Accompanying the above-mentioned topics, this review examines: (1) mtDNA maintenance in human health and disease, (2) reports of DNA polymerases theta and zeta (Rev3) localizing to mitochondria, and (3) additional drugs with off-target effects on mitochondrial function. Lastly, mtDNA damage may induce cell death; therefore, the possibility of utilizing compounds that disrupt mtDNA maintenance to kill cancer cells is discussed.