The AAA+ protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization

Many copies of mammalian mitochondrial DNA contain a short triple-stranded region, or displacement loop (D-loop), in the major noncoding region. In the 35 years since their discovery, no function has been assigned to mitochondrial D-loops. We purified mitochondrial nucleoprotein complexes from rat liver and identified a previously uncharacterized protein, ATAD3p. Localization studies suggested that human ATAD3 is a component of many, but not all, mitochondrial nucleoids. Gene silencing of ATAD3 by RNA interference altered the structure of mitochondrial nucleoids and led to the dissociation of mitochondrial DNA fragments held together by protein, specifically, ones containing the D-loop region. In vitro, a recombinant fragment of ATAD3p bound to supercoiled DNA molecules that contained a synthetic D-loop, with a marked preference over partially relaxed molecules with a D-loop or supercoiled DNA circles. These results suggest that mitochondrial D-loops serve to recruit ATAD3p for the purpose of forming or segregating mitochondrial nucleoids.

[1]  D. Bogenhagen,et al.  Human Mitochondrial DNA Nucleoids Are Linked to Protein Folding Machinery and Metabolic Enzymes at the Mitochondrial Inner Membrane* , 2006, Journal of Biological Chemistry.

[2]  Robert D. Finn,et al.  Pfam: clans, web tools and services , 2005, Nucleic Acids Res..

[3]  R. A. Butow,et al.  The organization and inheritance of the mitochondrial genome , 2005, Nature Reviews Genetics.

[4]  H. Jacobs,et al.  A bidirectional origin of replication maps to the major noncoding region of human mitochondrial DNA. , 2005, Molecular cell.

[5]  Joanna Poulton,et al.  Detection of mitochondrial DNA depletion in living human cells using PicoGreen staining. , 2005, Experimental cell research.

[6]  B. Kaufman,et al.  Aconitase Couples Metabolic Regulation to Mitochondrial DNA Maintenance , 2005, Science.

[7]  N. Ashley,et al.  Twinkle helicase is essential for mtDNA maintenance and regulates mtDNA copy number. , 2004, Human molecular genetics.

[8]  Marjan S. Bolouri,et al.  Integrated Analysis of Protein Composition, Tissue Diversity, and Gene Regulation in Mouse Mitochondria , 2003, Cell.

[9]  P. Perlman,et al.  A function for the mitochondrial chaperonin Hsp60 in the structure and transmission of mitochondrial DNA nucleoids in Saccharomyces cerevisiae , 2003, The Journal of cell biology.

[10]  R. Kobayashi,et al.  Protein Components of Mitochondrial DNA Nucleoids in Higher Eukaryotes* , 2003, Molecular & Cellular Proteomics.

[11]  D. Wigley,et al.  Multiple roles for ATP hydrolysis in nucleic acid modifying enzymes , 2003, The EMBO journal.

[12]  A. M. van der Bliek,et al.  Composition and dynamics of human mitochondrial nucleoids. , 2003, Molecular biology of the cell.

[13]  N. Hamasaki,et al.  Human mitochondrial DNA is packaged with TFAM. , 2003, Nucleic acids research.

[14]  H. Jacobs,et al.  Biased Incorporation of Ribonucleotides on the Mitochondrial L-Strand Accounts for Apparent Strand-Asymmetric DNA Replication , 2002, Cell.

[15]  D. Kamashev,et al.  The Bacterial Histone-like Protein HU Specifically Recognizes Similar Structures in All Nucleic Acids , 2002, The Journal of Biological Chemistry.

[16]  G. Comi,et al.  Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria , 2001, Nature Genetics.

[17]  C. Slaughter,et al.  In organello formaldehyde crosslinking of proteins to mtDNA: identification of bifunctional proteins. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[18]  D. MacAlpine,et al.  The numbers of individual mitochondrial DNA molecules and mitochondrial DNA nucleoids in yeast are co‐regulated by the general amino acid control pathway , 2000, The EMBO journal.

[19]  D. Turnbull,et al.  Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA , 1999, Nature Genetics.

[20]  T. Megraw,et al.  Functional complementarity between the HMG1-like yeast mitochondrial histone HM and the bacterial histone-like protein HU. , 1993, The Journal of biological chemistry.

[21]  W. L. Fangman,et al.  The localization of replication origins on ARS plasmids in S. cerevisiae , 1987, Cell.

[22]  D. Lilley,et al.  The genetic control of DNA supercoiling in Salmonella typhimurium. , 1984, The EMBO journal.

[23]  D. A. Clayton,et al.  Replication of animal mitochondrial DNA , 1982, Cell.

[24]  T. Shibata,et al.  Homologous pairing in genetic recombination: formation of D loops by combined action of recA protein and a helix-destabilizing protein. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[25]  H. Kasamatsu,et al.  A novel closed-circular mitochondrial DNA with properties of a replicating intermediate. , 1971, Proceedings of the National Academy of Sciences of the United States of America.

[26]  A. Arnberg,et al.  The presence of DNA molecules with a displacement loop in standard mitochondrial DNA preparations. , 1971, Biochimica et biophysica acta.

[27]  B. J. Brewer,et al.  Analysis of replication intermediates by two-dimensional agarose gel electrophoresis. , 1995, Methods in enzymology.