Mechanisms of Human Mitochondrial DNA Maintenance: The Determining Role of Primary Sequence and Length over Function

Although the regulation of mitochondrial DNA (mtDNA) copy number is performed by nuclear-coded factors, very little is known about the mechanisms controlling this process. We attempted to introduce nonhuman ape mtDNA into human cells harboring either no mtDNA or mutated mtDNAs (partial deletion and tRNA gene point mutation). Unexpectedly, only cells containing no mtDNA could be repopulated with nonhuman ape mtDNA. Cells containing a defective human mtDNA did not incorporate or maintain ape mtDNA and therefore died under selection for oxidative phosphorylation function. On the other hand, foreign human mtDNA was readily incorporated and maintained in these cells. The suicidal preference for self-mtDNA showed that functional parameters associated with oxidative phosphorylation are less relevant to mtDNA maintenance and copy number control than recognition of mtDNA self-determinants. Non–self-mtDNA could not be maintained into cells with mtDNA even if no selection for oxidative phosphorylation was applied. The repopulation kinetics of several mtDNA forms after severe depletion by ethidium bromide treatment showed that replication and maintenance of mtDNA in human cells are highly dependent on molecular features, because partially deleted mtDNA molecules repopulated cells significantly faster than full-length mtDNA. Taken together, our results suggest that mtDNA copy number may be controlled by competition for limiting levels of trans-acting factors that recognize primarily mtDNA molecular features. In agreement with this hypothesis, marked variations in mtDNA levels did not affect the transcription of nuclear-coded factors involved in mtDNA replication.

Stimulation of human 8-oxoguanine-DNA glycosylase by AP-endonuclease: potential coordination of the initial steps in base excision repair

8-Oxoguanine-DNA glycosylase 1 (OGG1), with intrinsic AP lyase activity, is the major enzyme for repairing 7,8-dihydro-8-oxoguanine (8-oxoG), a critical mutagenic DNA lesion induced by reactive oxygen species. Human OGG1 excised the damaged base from an 8-oxoG·C-containing duplex oligo with a very low apparent kcat of 0.1 min–1 at 37°C and cleaved abasic (AP) sites at half the rate, thus leaving abasic sites as the major product. Excision of 8-oxoG by OGG1 alone did not follow Michaelis–Menten kinetics. However, in the presence of a comparable amount of human AP endonuclease (APE1) the specific activity of OGG1 was increased ∼5-fold and Michaelis–Menten kinetics were observed. Inactive APE1, at a higher molar ratio, and a bacterial APE (Nfo) similarly enhanced OGG1 activity. The affinity of OGG1 for its product AP·C pair (Kd ∼ 2.8 nM) was substantially higher than for its substrate 8-oxoG·C pair (Kd ∼ 23.4 nM) and the affinity for its final β-elimination product was much lower (Kd ∼ 233 nM). These data, as well as single burst kinetics studies, indicate that the enzyme remains tightly bound to its AP product following base excision and that APE1 prevents its reassociation with its product, thus enhancing OGG1 turnover. These results suggest coordinated functions of OGG1 and APE1, and possibly other enzymes, in the DNA base excision repair pathway.

Overexpression of a Rrp1 transgene reduces the somatic mutation and recombination frequency induced by oxidative DNA damage in Drosophila melanogaster.

Recombination repair protein 1 (Rrp1) includes a C-terminal region homologous to several DNA repair proteins, including Escherichia coli exonuclease III and human APE, that repair oxidative and alkylation damage to DNA. The nuclease activities of Rrp1 include apurinic/apyrimidinic endonuclease, 3'-phosphodiesterase, 3'-phosphatase, and 3'-exonuclease. As shown previously, the C-terminal nuclease region of Rrp1 is sufficient to repair oxidative- and alkylation-induced DNA damage in repair-deficient E. coli mutants. DNA strand-transfer and single-stranded DNA renaturation activities are associated with the unique N-terminal region of Rrp1, which suggests possible additional functions that include recombinational repair or homologous recombination. By using the Drosophila w/w+ mosaic eye system, which detects loss of heterozygosity as changes in eye pigmentation, somatic mutation and recombination frequencies were determined in transgenic flies overexpressing wild-type Rrp1 protein from a heat-shock-inducible transgene. A large decrease in mosaic clone frequency is observed when Rrp1 overexpression precedes treatment with gamma-rays, bleomycin, or paraquat. In contrast, Rrp1 overexpression does not alter the spot frequency after treatment with the alkylating agents methyl methanesulfonate or methyl nitrosourea. A reduction in mosaic clone frequency depends on the expression of the Rrp1 transgene and on the nature of the induced DNA damage. These data suggest a lesion-specific involvement of Rrp1 in the repair of oxidative DNA damage.

Phosphatidylinositol 3-kinase signals activation of p70 S6 kinase in situ through site-specific p70 phosphorylation.

The p70 S6 kinase is activated by insulin and mitogens through multisite phosphorylation of the enzyme. One set of activating phosphorylations occurs in a putative autoinhibitory domain in the noncatalytic carboxyl-terminal tail. Deletion of this tail yields a variant (p70 delta CT104) that nevertheless continues to be mitogen regulated. Coexpression with a recombinant constitutively active phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) gives substantial activation of both full-length p70 and p70 delta CT104 but not Rsk. Activation of p70 delta CT104 by PI 3-kinase and inhibition by wortmannin are each accompanied by parallel and selective changes in the phosphorylation of p70 Thr-252. A Thr or Ser at this site, in subdomain VIII of the catalytic domain just amino-terminal to the APE motif, is necessary for p70 40S kinase activity. The inactive ATP-binding site mutant K123M p70 delta CT104 undergoes phosphorylation of Thr-252 in situ but does not undergo direct phosphorylation by the active PI 3-kinase in vitro. PI 3-kinase provides a signal necessary for the mitogen activation of the p70 S6 kinase, which directs the site-specific phosphorylation of Thr-252 in the p70 catalytic domain, through a distinctive signal transduction pathway.

Fervor; poemas.

"Apéndice" (12 folded pages at end) containing poems possibly omitted in compilation of original text. Noticia de Porfirio Martínez Peñaloza. 1967?

Forense: un auto de la Corte Suprema de la República, en la cuestión Banco Nacional de Bolivia y don Inocencio Balanza, ante el fallo de la opinión pública.

"Apéndices [documentales]": p. 1-L at end.

Obras políticas de Bernardo Monteagudo.

Noticia preliminar, por R. Rojas.--libro I. Memoria política.--libro II. Federación americana.--libro III. Propaganda revolucionaria.--libro IV. Exposición de tareas.--libro V. Discursos patrióticos.--libro VI. Epistolario.--Apéndice: Artículos de "El Independiente" atibuídos a Monteagudo (Buenos Aires, 1815)

Determinación del territorio de la República del Ecuador confinante con el de la República del Perú; dictamen en derecho

"Apéndice a. Fuentes especiales de información" : p. [381]-386.

Obras completas /

Includes bibliographical references.

Apéndice al diccionario histórico, y forense del Derecho Real de España : Tomo segundo /

Sign.: a8, A-Z8, 2A-2O8, 2P6.

Apuntes de libros y foiletos impresos en España y el extranjero que tratan expresamente de Cuba desde principios del siglo xvii hasta 1812 y de las dispociones de gobierno ímpresas en la Habana desde 1753 hasta 1800.

Appendix is separately paged and has separate t.-p.: Apéndices, Impresos de la Real sociedad patriótica y del Real consulado de la Habana. Adiciones á La imprenta en la Habana de José Toribio Medina. Habana, Imp. de "Cuba y América, " 1907. (16p.)

Galeria biografica de los generales de marina, jefes y personajes notables que figuraron en la misma corporacion desde 1700 á 1868

Imprint varies: v. 3, Impr. de F. Garcia y ca.; v. [4] Impr. de F. Garcia y D. Caravera.

Juicio imparcial sobre las letres, en forma de breve, que ha publicado la Curia Romana, en que se intentan derogar ciertos edictos del serenís imo señor infante duque de Parma, y disputarle la soberanía temporal con este pretexto.

A criticism of "litterae in forma brevis quibus abrogantur nonnulla edicta in ducatu parmensi et placentino edita, libertati immunitati et jurisdictioni ecclesiasticae praejudicialia, Romae, 1768." cf. Lottici-Sitti, Bibliografia generale per la storia parmense, Parma, 1904 (no. 1025-1039) in which only the French translation of the present work is quoted (no. 1038)

Patriarchikoi pinakes : eidēseis historikai viographikai peri tōn patriarchōn Kōnstantinoupoleōs apo Andreou tou Prōtoklētou mechris Iōakeim Gʹ tou apo Thessalonikēs, 36-1884 /

Projected supplements and alphabetical index, mentioned in footnote, p. 709, were never published.

Opere di Pietro Metastasio.

Vol. 16 includes general index (p. [361]-366)