Localization of mitochondrial DNA base excision repair to an inner membrane-associated particulate fraction.
Mitochondrial DNA (mtDNA) contains high levels of oxidative damage relative to nuclear DNA. A full, functional DNA base excision repair (BER) pathway is present in mitochondria, to repair oxidative DNA lesions. However, little is known about the organization of this pathway within mitochondria. Here, we provide evidence that the mitochondrial BER proteins are not freely soluble, but strongly associated with an inner membrane-containing particulate fraction. Uracil DNA glycosylase, oxoguanine DNA glycosylase and DNA polymerase gamma activities all co-sedimented with this particulate fraction and were not dissociated from it by detergent (0.1% or 1.0% NP40) treatment. The particulate associations of these activities were not due to their binding mtDNA, which is itself associated with the inner membrane, as they also localized to the particulate fraction of mitochondria from 143B (TK-) rho(0) cells, which lack mtDNA. However, all of the BER activities were at least partially solubilized from the particulate fraction by treatment with 150-300 mM NaCl, suggesting that electrostatic interactions are involved in the association. The biological implications of the apparent immobilization of BER proteins are discussed.
Stuart
J A
JA
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Box 1, Baltimore, MD 21224, USA.
Mayard
S
S
Hashiguchi
K
K
Souza-Pinto
N C
NC
Bohr
V A
VA
D002458:Cell Fractionation; D002460:Cell Line; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000032:analysis* / Q000302:isolation & purification; D004272:DNA, Mitochondrial / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D024101:Mitochondrial Proteins / Q000032:analysis* / Q000302:isolation & purification; D012995:Solubility; D055672:Static Electricity
D004272:DNA, Mitochondrial; D024101:Mitochondrial Proteins; D045643:DNA Repair Enzymes