In higher eukaryotes, mitochondria play multiple roles in energy production, signaling, and biosynthesis. Mitochondria possess multiple copies of mitochondrial DNA (mtDNA), which encodes 37 genes that are essential for mitochondrial and cellular function. When mtDNA is challenged by endogenous and exogenous factors, mtDNA undergoes repair, degradation, and compensatory synthesis. mtDNA degradation is an emerging pathway in mtDNA damage response and maintenance. A key factor involved is the human mitochondrial genome maintenance exonuclease 1 (MGME1). Despite previous biochemical and functional studies, controversies exist regarding the polarity of MGME1-mediated DNA cleavage. Also, how DNA sequence may affect the activities of MGME1 remains elusive. Such information is not only fundamental to the understanding of MGME1 but critical for deciphering the mechanism of mtDNA degradation. Herein, we use quantitative assays to examine the effects of substrate structure and sequence on the DNA-binding and enzymatic activities of MGME1. We demonstrate that MGME1 binds to and cleaves from the 5′-end of single-stranded DNA substrates, especially in the presence of 5′-phosphate, which plays an important role in DNA binding and optimal cleavage by MGME1. In addition, MGME1 tolerates certain modifications at the terminal end, such as a 5′-deoxyribosephosphate intermediate formed in base excision repair. We show that MGME1 processes different sequences with varying efficiencies, with dT and dC sequences being the most and least efficiently digested, respectively. Our results provide insights into the enzymatic properties of MGME1 and a rationale for the coordination of MGME1 with the 3′–5′ exonuclease activity of DNA polymerase γ in mtDNA degradation.

在高等真核生物中，线粒体在能量产生、信号传导和生物合成中发挥多重作用。线粒体拥有多个拷贝的线粒体 DNA (mtDNA)，它编码 37 个对线粒体和细胞功能至关重要的基因。当mtDNA受到内源性和外源性因素的挑战时，mtDNA会经历修复、降解和补偿合成。mtDNA 降解是 mtDNA 损伤反应和维持的新兴途径。涉及的一个关键因素是人类线粒体基因组维持核酸外切酶 1 (MGME1)。尽管先前进行了生化和功能研究，但关于 MGME1 介导的 DNA 切割的极性仍存在争议。此外，DNA 序列如何影响 MGME1 的活性仍然难以捉摸。这些信息不仅是理解 MGME1 的基础，而且对于破译 mtDNA 降解的机制至关重要。在这里，我们使用定量分析来检查底物结构和序列对 MGME1 的 DNA 结合和酶活性的影响。我们证明 MGME1 与单链 DNA 底物的 5' 端结合并从其切割，特别是在 5'-磷酸盐存在的情况下，这在 MGME1 的 DNA 结合和最佳切割中起重要作用。此外，MGME1 耐受末端的某些修饰，例如在碱基切除修复中形成的 5'-脱氧核糖磷酸中间体。我们表明 MGME1 以不同的效率处理不同的序列，其中 dT 和 dC 序列分别是最有效和最不有效的消化。