Published online 23 April 2004
Nucleic Acids Research, 2004, Vol. 32, No. 7 2181-2192
© 2004 Oxford University Press
DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from cells lacking mitochondrial DNA
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA and 1 Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, PO Box 12233, 111TW Alexander Drive, Research Triangle Park, NC 27709, USA
*To whom correspondence should be addressed at present address: 5600 Nathan Shock Dr., Box 1, Baltimore, MD 21224, USA. Tel: +1 410 558 8332; Fax: +1 410 558 8157; Email: bohrv{at}grc.nia.nih.gov
Present address:
J.A. Stuart, Department of Biology, Brock University, St Catharines, Ontario, Canada L2S 3A1
Received December 31, 2003; Revised and Accepted March 21, 2004
Mitochondrial DNA (mtDNA) contains higher steady-state levels of oxidative damage and mutates at rates significantly greater than nuclear DNA. Oxidative lesions in mtDNA are removed by a base excision repair (BER) pathway. All mtDNA repair proteins are nuclear encoded and imported. Most mtDNA repair proteins so far discovered are either identical to nuclear DNA repair proteins or isoforms of nuclear proteins arising from differential splicing. Regulation of mitochondrial BER is therefore not expected to be independent of nuclear BER, though the extent to which mitochondrial BER is regulated with respect to mtDNA amount or damage is largely unknown. Here we have measured DNA BER activities in lysates of mitochondria isolated from human 143B TK– osteosarcoma cells that had been depleted of mtDNA (
0) or not (wt). Despite the total absence of mtDNA in the 0 cells, a complete mitochondrial BER pathway was present, as demonstrated using an in vitro assay with synthetic oligonucleotides. Measurement of individual BER protein activities in mitochondrial lysates indicated that some BER activities are insensitive to the lack of mtDNA. Uracil and 8-oxoguanine DNA glycosylase activities were relatively insensitive to the absence of mtDNA, only about 25% reduced in 0 relative to wt cells. Apurinic/apyrimidinic (AP) endonuclease and polymerase 
activities were more affected, 65 and 45% lower, respectively, in 0 mitochondria. Overall BER activity in lysates was also about 65% reduced in 0 mitochondria. To identify the limiting deficiencies in BER of 0 mitochondria we supplemented the BER assay of mitochondrial lysates with pure uracil DNA glycosylase, AP endonuclease and/or the catalytic subunit of polymerase . BER activity was stimulated by addition of uracil DNA glycosylase and polymerase . However, no addition or combination of additions stimulated BER activity to wt levels. This suggests that an unknown activity, factor or interaction important in BER is deficient in 0 mitochondria. While nuclear BER protein levels and activities were generally not altered in 0 cells, AP endonuclease activity was substantially reduced in nuclear and in whole cell extracts. This appeared to be due to reduced endogenous reactive oxygen species (ROS) production in 0 cells, and not a general dysfunction of 0 cells, as exposure of cells to ROS rapidly stimulated increases in AP endonuclease activities and APE1 protein levels.
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