Souza-Pinto N C, Croteau D L, Hudson E K, Hansford R G, Bohr V A
Laboratory of Molecular Genetics, Box 1, National Institute on Aging, National Institutes of Health,5600 Nathan Shock Drive, Baltimore, MD 21224, USA.
Nucleic Acids Res. 1999 Apr 15;27(8):1935-42. doi: 10.1093/nar/27.8.1935.
The mitochondrial theory of aging postulates that organisms age due to the accumulation of DNA damage and mutations in the multiple mitochondrial genomes, leading to mitochondrial dysfunction. Among the wide variety of DNA damage, 8-oxo-deoxyguanosine (8-oxo-dG) has received the most attention due to its mutagenicity and because of the possible correlation between its accumulation and pathological processes like cancer, degenerative diseases and aging. Although still controversial, many studies show that 8-oxo-dG accumulates with age in the mitochondrial (mt) DNA. However, little is known about the processing of this lesion and no study has yet examined whether mtDNA repair changes with age. Here, we report the first study on age-related changes in mtDNA repair, accomplished by assessing the cleavage activity of mitochondrial extracts towards an 8-oxo-dG-containing substrate. In this study, mitochondria obtained from rat heart and liver were used. We find that this enzymatic activity is higher in 12 and 23 month-old rats than in 6 month-old rats, in both liver and heart extracts. These mitochondrial extracts also cleave oligonucleotides containing a U:A mismatch, at the uracil position, reflecting the combined action of mitochondrial uracil DNA glycosylase (mtUDG) and mitochondrial apurinic/apyrimidinic (AP) endonucleases. The mtUDG activity did not change with age in liver mitochondria, but there was a small increase in activity from 6 to 23 months in rat heart extracts, after normalization to citrate synthase activity. Endonuclease G activity, measured by a plasmid relaxation assay, did not show any age-associated change in liver, but there was a significant decrease from 6 to 23 months in heart mitochondria. Our results suggest that the mitochondrial capacity to repair 8-oxo-dG, the main oxidative base damage suggested to accumulate with age in mtDNA, does not decrease, but rather increases with age. The specific increase in 8-oxo-dG endonuclease activity, rather than a general up-regulation of DNA repair in mitochondria, suggests an induction of the 8-oxo-dG-specific repair pathway with age.
衰老的线粒体理论假定，生物体衰老是由于多个线粒体基因组中DNA损伤和突变的积累，导致线粒体功能障碍。在各种各样的DNA损伤中，8-氧代脱氧鸟苷（8-oxo-dG）因其致突变性以及其积累与癌症、退行性疾病和衰老等病理过程之间可能存在的关联而受到了最多关注。尽管仍存在争议，但许多研究表明，8-oxo-dG在线粒体（mt）DNA中会随着年龄的增长而积累。然而，关于这种损伤的处理知之甚少，而且尚无研究探讨mtDNA修复是否会随年龄变化。在此，我们报告了第一项关于mtDNA修复与年龄相关变化的研究，该研究通过评估线粒体提取物对含8-oxo-dG底物的切割活性来完成。在本研究中，使用了从大鼠心脏和肝脏获取的线粒体。我们发现，在肝脏和心脏提取物中，12月龄和23月龄大鼠的这种酶活性均高于6月龄大鼠。这些线粒体提取物还会在尿嘧啶位置切割含有U:A错配的寡核苷酸，这反映了线粒体尿嘧啶DNA糖基化酶（mtUDG）和线粒体脱嘌呤/脱嘧啶（AP）内切核酸酶的联合作用。肝脏线粒体中的mtUDG活性不会随年龄变化，但在将大鼠心脏提取物中的活性标准化为柠檬酸合酶活性后，6至23月龄时活性有小幅增加。通过质粒松弛测定法测量的核酸内切酶G活性在肝脏中未显示出任何与年龄相关的变化，但在心脏线粒体中从6至23月龄有显著下降。我们的结果表明，线粒体修复8-oxo-dG（这是一种被认为会随年龄在mtDNA中积累的主要氧化碱基损伤）的能力不会下降，反而会随年龄增加。8-oxo-dG内切核酸酶活性的特异性增加，而非线粒体中DNA修复的普遍上调，表明随着年龄增长，8-oxo-dG特异性修复途径被诱导。
