Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.
J Biol Chem. 2012 Oct 26;287(44):37259-68. doi: 10.1074/jbc.M112.389965. Epub 2012 Sep 4.
Mgm101 is a Rad52-type recombination protein of bacteriophage origin required for the repair and maintenance of mitochondrial DNA (mtDNA). It forms large oligomeric rings of ∼14-fold symmetry that catalyze the annealing of single-stranded DNAs in vitro. In this study, we investigated the structural elements that contribute to this distinctive higher order structural organization and examined its functional implications. A pair of vicinal cysteines, Cys-216 and Cys-217, was found to be essential for mtDNA maintenance. Mutations to the polar serine, the negatively charged aspartic and glutamic acids, and the hydrophobic amino acid alanine all destabilize mtDNA in vivo. The alanine mutants have an increased propensity of forming macroscopic filaments. In contrast, mutations to aspartic acid drastically destabilize the protein and result in unstructured aggregates with severely reduced DNA binding activity. Interestingly, the serine mutants partially disassemble the Mgm101 rings into smaller oligomers. In the case of the C216S mutant, a moderate increase in DNA binding activity was observed. By using small angle x-ray scattering analysis, we found that Mgm101 forms rings of ∼200 Å diameter in solution, consistent with the structure previously established by transmission electron microscopy. We also found that the C216A/C217A double mutant tends to form broken rings, which likely provide free ends for seeding the growth of the super-stable but functionally defective filaments. Taken together, our data underscore the importance of a delicately maintained ring structure critical for Mgm101 activity. We discuss a potential role of Cys-216 and Cys-217 in regulating Mgm101 function and the repair of damaged mtDNA under stress conditions.
Mgm101 是一种源自噬菌体的 Rad52 型重组蛋白，对于线粒体 DNA（mtDNA）的修复和维持是必需的。它形成约 14 倍对称的大型寡聚环，体外催化单链 DNA 的退火。在这项研究中，我们研究了有助于这种独特的高级结构组织的结构元素，并研究了其功能意义。发现一对相邻的半胱氨酸，Cys-216 和 Cys-217，对于 mtDNA 的维持是必需的。对极性丝氨酸、带负电荷的天冬氨酸和谷氨酸以及疏水性氨基酸丙氨酸的突变都会使 mtDNA 在体内不稳定。丙氨酸突变体形成宏观细丝的倾向增加。相比之下，天冬氨酸突变严重破坏蛋白质并导致无结构聚集体，其 DNA 结合活性严重降低。有趣的是，丝氨酸突变体部分地将 Mgm101 环分解成较小的寡聚物。在 C216S 突变体的情况下，观察到 DNA 结合活性适度增加。通过使用小角度 X 射线散射分析，我们发现 Mgm101 在溶液中形成直径约为 200Å 的环，与先前通过透射电子显微镜建立的结构一致。我们还发现 C216A/C217A 双突变体倾向于形成断裂的环，这可能为超稳定但功能缺陷的细丝的生长提供自由端。总之，我们的数据强调了维持 Mgm101 活性的精细环结构的重要性。我们讨论了 Cys-216 和 Cys-217 在调节 Mgm101 功能和在应激条件下修复受损 mtDNA 中的潜在作用。
