Structure and Nano-Mechanics of DNA during the Initial Stages of Methyl-Directed Mismatch Repair
Structure and Nano-Mechanics of DNA during the Initial Stages of Methyl-Directed Mismatch Repair
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甲基定向错配修复初始阶段 DNA 的结构和纳米力学
DOI：
10.1016/j.bpj.2014.11.406
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发表时间：
2015
影响因子：
3.4
通讯作者：
Marszalek, Piotr E.
中科院分区：
文献类型：
--
作者：
Josephs, Eric A.;Marszalek, Piotr E.
转录偶联修复（TCR）是核苷酸切除修复（NER）的一个子途径，已知其比整体NER修复（GGR）更有效。在这里，我们使用单个DNA分子的磁性捕获来研究TCR蛋白与停滞的RNA聚合酶（RNAP）的相互作用。使用位于转录链上的无CTP盒或胸腺嘧啶-胸腺嘧啶二聚体将单个RNAP分子固定在DNA转录物上。停滞的RNAP被Mfd移位酶取代，并形成长寿命的中间体。我们的特点之间的相互作用UvrA/UvrB蛋白质和长寿命的中间体形成后，MFD位移RNAP。这种相互作用导致形成切割前复合物，该复合物对DNA切割具有催化活性，在加入UvrC. 342-Pos Board B122 Investigation of the Tus-ter Blocking Efficacy during the Chromosome Replication of Live Escherichia Coli Cells后，在磁阱中测试该复合物的活性Sriram Tiruvadi Krishnan，M.放大图片创作者：John W.戴克生物纳米科学，德尔夫特理工大学，德尔夫特，荷兰。在大肠在大肠杆菌中，环状染色体通过在单个复制起点（oriC）组装的两个复制体复合物以双向方式复制。两个复制叉被认为终止于终止区，其两侧是10个ter位点。这些位点中的每一个都被终止利用物质（Tus）蛋白结合，从而形成Tus-特复合物。体外研究表明，当复制叉从非允许侧接近Tus-ter复合物时被阻断，而从允许侧则不被阻断。然而，Tus-ter复合物对从非允许侧接近的复制叉的阻断功效以及随后的动力学尚未在活细胞中进行检查。为了阐明这些过程在体内，我们利用定量荧光显微镜结合微流控研究四个不同的E。在大肠杆菌菌株中，具有正常oriC或仅具有异位拷贝（oriZ）（插入到大肠杆菌菌株中的344 kb处）。大肠杆菌遗传图谱沿着染色体），并且具有野生型Tus存在或敲除（Dtus）。在oriZ菌株中，顺时针复制叉（通过其滑动夹标记）比逆时针复制叉更早地遇到Tus-ter复合物（通过其非允许面）。在跟踪oriZ菌株中复制叉的进展时，我们发现Tus-ter复合物的存在降低了复制速率。通过监测叉的荧光强度以及染色体位点的复制，我们可以确定复制叉仍然可以绕过Tustercomplex的非允许侧。TheoriZ-Dtus菌株表现出与oriC菌株相似的复制体动力学，而oriZ基因座主要向细胞极移动的染色体动力学则有很大不同。
Transcription-coupled repair (TCR), a subpathway of nucleotide excision repair (NER), has been known to lead to more efficient repair than the global NER repair (GGR). Here we use magnetic trapping of single DNA molecules to study the interactions of TCR proteins with stalled RNA polymerase (RNAP). Single RNAP molecules are stalled on a DNA transcript using either a CTP-less cassette or a thymine-thymine dimer located on the transcribed strand. Stalled RNAP is displaced by the Mfd translocase, and a long-lived intermediate is formed. We characterize the interaction between UvrA/UvrB proteins and the long-lived intermediate formed upon Mfd displacement of RNAP. This interaction leads to formation of a pre-incision complex that is catalytically competent for DNA incision, and the activity of this complex is tested for in the magnetic trap upon addition of UvrC.342-Pos Board B122 Investigation of the Tus-ter Blocking Efficacy during the Chromosome Replication of Live Escherichia Coli Cells Sriram Tiruvadi Krishnan, M. Charl Moolman, Roy de Leeuw, Jacob WJ Kerssemakers, Nynke H. Dekker. Bionanoscience, Delft University of Technology, Delft, Netherlands. In E. coli, a circular chromosome is replicated in a bi-directional manner by two replisome complexes that assemble at a single origin of replication (oriC). The two replication forks are thought to terminate in the termination region, which is flanked by 10 ter sites. Each of these sites is bound by a Termination utilization substance (Tus) protein, thereby forming Tus-tercomplexes. In vitrostudies have shown that the replication forks are blocked when they approach a Tus-ter complex from the non-permissive side, but not from the permissive side. However, the blocking efficacy of the Tus-ter complex on a replication fork approaching from the non-permissive side, and the subsequent dynamics, have not been examined in live cells. To shed light on these processes in vivo, we utilize quantitative fluorescence microscopy combined with microfluidics to study four different E. coli strains that possess either a normal oriC or only an ectopic copy (oriZ)(inserted 344 kb in the E. coli genetic map along the chromosome), and have either wildtype Tus present or knocked-out (Dtus). In the oriZ strain, the clockwise replication fork (labelled via its sliding clamps) encounters the Tus-ter complex (via its non-permissive face) earlier than the counter-clockwise fork does. On tracking the progression of the replication forks in oriZ strain, we find that the presence of the Tus-ter complex reduces the rate of replication. By monitoring the fluorescence intensity of the fork as well as the duplication of a chromosomal locus, we can determine that a replication fork can nonetheless bypass the nonpermissive side of a Tus-tercomplex. TheoriZ-Dtusstrain exhibits replisome dynamics similar to that of oriC strain, whereas the chromosome dynamics differ substantially where the oriZ locus moves mostly towards cell-pole.