Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Baltimore, MD 21224, United States.
DNA Repair (Amst). 2010 Oct 5;9(10):1080-9. doi: 10.1016/j.dnarep.2010.07.009. Epub 2010 Aug 23.
Mitochondrial transcription factor A (TFAM) is an essential component of mitochondrial nucleoids. TFAM plays an important role in mitochondrial transcription and replication. TFAM has been previously reported to inhibit nucleotide excision repair (NER) in vitro but NER has not yet been detected in mitochondria, whereas base excision repair (BER) has been comprehensively characterized in these organelles. The BER proteins are associated with the inner membrane in mitochondria and thus with the mitochondrial nucleoid, where TFAM is also situated. However, a function for TFAM in BER has not yet been investigated. This study examines the role of TFAM in BER. In vitro studies with purified recombinant TFAM indicate that it preferentially binds to DNA containing 8-oxoguanines, but not to abasic sites, uracils, or a gap in the sequence. TFAM inhibited the in vitro incision activity of 8-oxoguanine DNA glycosylase (OGG1), uracil-DNA glycosylase (UDG), apurinic endonuclease 1 (APE1), and nucleotide incorporation by DNA polymerase γ (pol γ). On the other hand, a DNA binding-defective TFAM mutant, L58A, showed less inhibition of BER in vitro. Characterization of TFAM knockdown (KD) cells revealed that these lysates had higher 8oxoG incision activity without changes in αOGG1 protein levels, TFAM KD cells had mild resistance to menadione and increased damage accumulation in the mtDNA when compared to the control cells. In addition, we found that the tumor suppressor p53, which has been shown to interact with and alter the DNA binding activity of TFAM, alleviates TFAM-induced inhibition of BER proteins. Together, the results suggest that TFAM modulates BER in mitochondria by virtue of its DNA binding activity and protein interactions.
线粒体转录因子 A（TFAM）是线粒体核体的重要组成部分。TFAM 在线粒体转录和复制中发挥重要作用。先前有报道称 TFAM 在体外抑制核苷酸切除修复（NER），但线粒体中尚未检测到 NER，而碱基切除修复（BER）已在这些细胞器中得到全面描述。BER 蛋白与线粒体的内膜相关，因此与线粒体核体相关，TFAM 也位于其中。然而，TFAM 在 BER 中的作用尚未被研究。本研究探讨了 TFAM 在 BER 中的作用。体外研究表明，纯化的重组 TFAM 优先结合含有 8-氧鸟嘌呤的 DNA，但不结合无碱基位点、尿嘧啶或序列中的缺口。TFAM 抑制 8-氧鸟嘌呤 DNA 糖基化酶（OGG1）、尿嘧啶 DNA 糖基化酶（UDG）、脱嘌呤内切酶 1（APE1）和 DNA 聚合酶 γ（pol γ）的体外切口活性。另一方面，DNA 结合缺陷型 TFAM 突变体 L58A 在体外对 BER 的抑制作用较小。TFAM 敲低（KD）细胞的特征表明，这些裂解物具有更高的 8oxoG 切口活性，而αOGG1 蛋白水平没有变化，与对照细胞相比，TFAM KD 细胞对 menadione 的抗性较弱，并且 mtDNA 中的损伤积累增加。此外，我们发现肿瘤抑制因子 p53 已被证明与 TFAM 相互作用并改变其 DNA 结合活性，可减轻 TFAM 诱导的 BER 蛋白抑制。总之，这些结果表明，TFAM 通过其 DNA 结合活性和蛋白相互作用来调节线粒体中的 BER。
