DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from cells lacking mitochondrial DNA.
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 (rho(0)) or not (wt). Despite the total absence of mtDNA in the rho(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 rho(0) relative to wt cells. Apurinic/apyrimidinic (AP) endonuclease and polymerase gamma activities were more affected, 65 and 45% lower, respectively, in rho(0) mitochondria. Overall BER activity in lysates was also about 65% reduced in rho(0) mitochondria. To identify the limiting deficiencies in BER of rho(0) mitochondria we supplemented the BER assay of mitochondrial lysates with pure uracil DNA glycosylase, AP endonuclease and/or the catalytic subunit of polymerase gamma. BER activity was stimulated by addition of uracil DNA glycosylase and polymerase gamma. 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 rho(0) mitochondria. While nuclear BER protein levels and activities were generally not altered in rho(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 rho(0) cells, and not a general dysfunction of rho(0) cells, as exposure of cells to ROS rapidly stimulated increases in AP endonuclease activities and APE1 protein levels.
Stuart
J A
JA
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
Hashiguchi
K
K
Wilson
D M
DM
Copeland
W C
WC
Souza-Pinto
N C
NC
Bohr
V A
VA
D020137:Base Pair Mismatch; D002457:Cell Extracts; D045744:Cell Line, Tumor; D002950:Citrate (si)-Synthase / Q000235:genetics / Q000378:metabolism; D045647:DNA Glycosylases / Q000378:metabolism*; D000074002:DNA Polymerase gamma; D004260:DNA Repair; D004272:DNA, Mitochondrial / Q000235:genetics; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D051981:Uracil-DNA Glycosidase
D002457:Cell Extracts; D004272:DNA, Mitochondrial; D006861:Hydrogen Peroxide; D002950:Citrate (si)-Synthase; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D045647:DNA Glycosylases; D051981:Uracil-DNA Glycosidase; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase