Physics Department, North Carolina State University, Raleigh, North Carolina, NC 27695, USA.
Center for Human Health and the Environment, North Carolina State University, Raleigh, North Carolina, NC 27695, USA.
Nucleic Acids Res. 2018 Nov 30;46(21):11287-11302. doi: 10.1093/nar/gky875.
Improper maintenance of the mitochondrial genome progressively disrupts cellular respiration and causes severe metabolic disorders commonly termed mitochondrial diseases. Mitochondrial single-stranded DNA binding protein (mtSSB) is an essential component of the mtDNA replication machinery. We utilized single-molecule methods to examine the modes by which human mtSSB binds DNA to help define protein interactions at the mtDNA replication fork. Direct visualization of individual mtSSB molecules by atomic force microscopy (AFM) revealed a random distribution of mtSSB tetramers bound to extended regions of single-stranded DNA (ssDNA), strongly suggesting non-cooperative binding by mtSSB. Selective binding to ssDNA was confirmed by AFM imaging of individual mtSSB tetramers bound to gapped plasmid DNA substrates bearing defined single-stranded regions. Shortening of the contour length of gapped DNA upon binding mtSSB was attributed to DNA wrapping around mtSSB. Tracing the DNA path in mtSSB-ssDNA complexes with Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy established a predominant binding mode with one DNA strand winding once around each mtSSB tetramer at physiological salt conditions. Single-molecule imaging suggests mtSSB may not saturate or fully protect single-stranded replication intermediates during mtDNA synthesis, leaving the mitochondrial genome vulnerable to chemical mutagenesis, deletions driven by primer relocation or other actions consistent with clinically observed deletion biases.
线粒体基因组的维护不当会逐渐破坏细胞呼吸，并导致严重的代谢紊乱，通常被称为线粒体疾病。线粒体单链 DNA 结合蛋白 (mtSSB) 是 mtDNA 复制机制的重要组成部分。我们利用单分子方法研究了人 mtSSB 结合 DNA 的方式，以帮助定义 mtDNA 复制叉处的蛋白质相互作用。原子力显微镜 (AFM) 对单个 mtSSB 分子的直接可视化显示，mtSSB 四聚体随机结合在单链 DNA (ssDNA) 的扩展区域，强烈表明 mtSSB 非协同结合。通过 AFM 对结合在带有定义单链区域的缺口质粒 DNA 底物上的单个 mtSSB 四聚体的成像，证实了对 ssDNA 的选择性结合。在结合 mtSSB 后，缺口 DNA 的轮廓长度缩短归因于 DNA 缠绕在 mtSSB 周围。用双共振频率增强静电力显微镜追踪 mtSSB-ssDNA 复合物中的 DNA 路径，在生理盐条件下建立了一种主要的结合模式，其中一条 DNA 链在每个 mtSSB 四聚体周围缠绕一次。单分子成像表明，mtSSB 可能不会在 mtDNA 合成过程中饱和或完全保护单链复制中间体，使线粒体基因组易受化学诱变、引物移位驱动的缺失或与临床观察到的缺失偏倚一致的其他作用的影响。
