Single-molecule DREEM imaging reveals DNA wrapping around human mitochondrial single-stranded DNA binding protein.
Single-molecule DREEM imaging reveals DNA wrapping around human mitochondrial single-stranded DNA binding protein.
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DOI：
10.1093/nar/gky875
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发表时间：
2018-11-30
影响因子：
14.9
通讯作者：
Copeland WC
中科院分区：
文献类型：
--
作者：
Kaur P;Longley MJ;Pan H;Wang H;Copeland WC
线粒体基因组的不适当维护逐渐破坏细胞呼吸并导致通常称为线粒体疾病的严重代谢紊乱。线粒体单链DNA结合蛋白（mtSSB）是线粒体DNA复制机制的重要组成部分。我们利用单分子方法来研究人类mtSSB结合DNA的模式，以帮助定义mtDNA复制叉处的蛋白质相互作用。通过原子力显微镜（AFM）直接观察单个mtSSB分子，发现与单链DNA（ssDNA）延伸区域结合的mtSSB四聚体的随机分布，强烈表明mtSSB的非合作结合。通过对与带有确定的单链区域的有缺口的质粒DNA底物结合的单个mtSSB四聚体进行原子力显微镜成像，证实了与ssDNA的选择性结合。结合mtSSB后，缺口DNA的轮廓长度缩短归因于DNA包裹在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，建立了在生理盐条件下一条DNA链围绕每个mtSSB四聚体缠绕一次的主要结合模式。单分子成像表明，在mtDNA合成过程中，mtSSB可能不会饱和或完全保护单链复制中间体，使线粒体基因组容易受到化学诱变、引物重新定位驱动的缺失或与临床观察到的缺失偏倚一致的其他行为的影响。
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.
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