The mitochondrial respiratory chain is a source of reactive oxygen species (ROS) that are responsible for oxidative modification of biomolecules, including proteins. Due to its association with mitochondrial DNA, DNA polymerase gamma (pol gamma) is in an environment to be oxidized by hydrogen peroxide and hydroxyl radicals that may be generated in the presence of iron ions associated with DNA. We tested whether human pol gamma was a possible target of ROS with H2O2 and investigated the effect on the polymerase activities and DNA binding efficiency. A 1 h treatment with 250 microM H2O2 significantly inhibited DNA polymerase activity of the p140 subunit and lowered its DNA binding efficiency. Addition of p55 to the p140 catalytic subunit prior to H2O2 treatment offered protection from oxidative inactivation. Oxidatively modified amino acid residues in pol gamma resulting from H2O2 treatment were observed in vitro as well as in vivo, in SV40-transfected human fibroblasts. Pol gamma was detected as one of the major oxidized mitochondrial matrix proteins, with a detectable decline in polymerase activity. These results suggest pol gamma as a target of oxidative damage, which may result in a reduction in mitochondrial DNA replication and repair capacities.
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP). This deficiency of TP leads to increased circulating levels of thymidine (deoxythymidine, dThd) and deoxyuridine (dUrd) and has been associated with multiple deletions and depletion of mitochondrial DNA (mtDNA). Here we describe 36 point mutations in mtDNA of tissues and cultured cells from MNGIE patients. Thirty-one mtDNA point mutations (86%) were T-to-C transitions, and of these, 25 were preceded by 5′-AA sequences. In addition, we identified a single base-pair mtDNA deletion and a TT-to-AA mutation. Next-nucleotide effects and dislocation mutagenesis may contribute to the formation of these mutations. These results provide the first demonstration that alterations of nucleoside metabolism can induce multiple sequence-specific point mutations in humans. We hypothesize that, in patients with TP deficiency, increased levels of dThd and dUrd cause mitochondrial nucleotide pool imbalances, which, in turn, lead to mtDNA abnormalities including site-specific point mutations
Peptide sequences obtained from the accessory subunit of Xenopus laevis mitochondrial DNA (mtDNA) polymerase ␥ (pol ␥) were used to clone the cDNA encoding this protein. Amino-terminal sequencing of the mitochondrial protein indicated the presence of a 44-amino-acid mitochondrial targeting sequence, leaving a predicted mature protein with 419 amino acids and a molecular mass of 47.3 kDa. This protein is associated with the larger, catalytic subunit in preparations of active mtDNA polymerase. The small subunit exhibits homology to its human, mouse, and Drosophila counterparts. Interestingly, significant homology to glycyl-tRNA synthetases from prokaryotic organisms reveals a likely evolutionary relationship. Since attempts to produce an enzymatically active recombinant catalytic subunit of Xenopus DNA pol ␥ have not been successful, we tested the effects of adding the small subunit of the Xenopus enzyme to the catalytic subunit of human DNA pol ␥ purified from baculovirus-infected insect cells. These experiments provide the first functional evidence that the small subunit of DNA pol ␥ stimulates processive DNA synthesis by the human catalytic subunit under physiological salt conditions. Mitochondrial DNA (mtDNA) is replicated by a DNA polymerase, DNA polymerase ␥ (pol ␥), that is distinct from nuclear DNA polymerases ␣, , ␦, ε, and . Since DNA pol ␥ represents only a small fraction of total cellular DNA polymerase, purification and characterization of the subunit composition of this enzyme have been difficult. Molecular cloning has contributed greatly to understanding the structure of DNA pol ␥ in different organisms. The catalytic subunits of DNA pol ␥ have been cloned for several organisms (6,16,17,27,34) and have been found to resemble family A of DNA polymerases, related to Escherichia coli DNA pol I. In Saccharomyces cerevisiae DNA pol ␥ is composed of a single polypeptide, while in Drosophila melanogaster DNA pol ␥ is comprised of two different polypeptides, a catalytic subunit of 125 kDa and an accessory subunit of 41 kDa (24, 33). The Drosophila subunits copurify and have been shown to interact, but the recombinant proteins have not yet been shown to be functional. The function of the small subunit, which we refer to as pol ␥B, is unknown. It has been proposed to influence the processivity of the catalytic subunit. Putative mammalian homologs of the Drosophila accessory subunit have been identified in sequence databases. One published purification scheme for human DNA pol ␥ suggested the existence of a small subunit (11), but the potential relationship between this polypeptide and Drosophila pol ␥B has not been established. Recently, the catalytic subunit of human DNA pol ␥ was expressed in an active form (10,19). Surprisingly, the recombinant catalytic subunit alone displayed most of the characteristics of the enzyme purified from human cells, which did not appear to contain a stoichiometric amount of a small subunit. Thus, it is not clear what role, if any, is played by putative human DNA pol ␥B.We hav...
For Abstract see ChemInform Abstract in Full Text.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.