DNA Triplexes and Replication

9723924 Mirkin这项研究将集中在DNA三链体在DNA复制中的作用。 在以前的研究中，我们发现在体外DNA聚合之前或期间形成的DNA三链体严重阻碍了这一过程。 这种现象的机制在于DNA聚合酶无法解开DNA模板中的三链体区域，而完全能够解开相应的双链体区域。 因此，DNA三链体可以在生理条件下形成对DNA聚合酶的稳定屏障。 我们推测，DNA三链体也可能抑制体内DNA复制。 支持这一假设，我们最近发现，一个特殊的三链体形成DNA序列，d（G）n（d（C）n，阻止复制叉运动在E。coli细胞。 这种效应取决于重复序列的长度及其相对于复制起点的方向，并且不会被蛋白质合成抑制剂消除。 这些结果表明d（G）n（d（C）n）在体内形成的一种不寻常的结构可能是导致复制停滞的原因。 本研究的具体目的是获得直接的证据，三链体结构的d（G）n（d（C）n重复序列导致复制停滞，并扩大这些研究，包括不同的三链体形成重复。 我们将完成d（G）n（d（C）n重复序列的详细突变分析，并结合复制中间体的分析，以区分该序列已知的众多结构机会。 为了直接了解这种重复序列在体内形成的结构，我们将在大肠杆菌中对含有（G）n（d（C）n）的质粒进行化学探测。大肠杆菌细胞使用结构特异性化学品氯乙醛。 我们还将分析不同长度和组成的三链体形成重复序列对大肠杆菌中质粒DNA复制的影响。大肠杆菌细胞使用复制中间体的二维电泳。 本研究的长期目标是了解形成三链体的DNA重复序列如何影响体内DNA复制的调节。 % DNA三链体结构符合生理条件下DNA复制的稳定屏障。 这项研究的长期目标是了解形成三链体的重复DNA序列如何影响活细胞中DNA复制的调节。 ***

9723924 Mirkin This research will focus on the role of DNA triplexes in DNA replication. In previous studies, we found that DNA triplexes formed prior to or during in vitro DNA polymerization severely block this process. The mechanism of this phenomenon lies in DNA polymerase's inability to untangle triplex regions in DNA templates, while being perfectly capable of untangling the corresponding duplex regions. Thus, DNA triplexes can form a stable barrier to DNA polymerases under physiological conditions. We hypothesize, that DNA triplexes might also inhibit DNA replication in vivo. Supporting this hypothesis, we recently found that a particular triplex-forming DNA sequence, d(G)n(d(C)n, blocks replication fork movement in E. coli cells. This effect depends on the length of the repeated sequence and its orientation relative to the replication origin, and is not abolished by a protein synthesis inhibitor. These results are very suggestive of an unusual structure formed by d(G)n(d(C)n in vivo that may be responsible for the replication arrest. The specific aim of this research is to obtain direct proof that the triplex structure of the d(G)n(d(C)n repeated sequence causes replication arrest, and to expand these studies to include different triplex-forming repeats. We will accomplish detailed mutational analysis of the d(G)n(d(C)n repeat combined with analysis of replication intermediates to distinguish between the numerous structural opportunities known for this sequence. To provide direct insight on the structure formed by this repeated sequence in vivo, we will carry out chemical probing of (G)n(d(C)n-containing plasmids in E. coli cells using the structure-specific chemical chloroacetaldehyde. We will also analyze the effects of triplex-forming repeated sequences of various length and composition on plasmid DNA replication in E. coli cells using two-dimensional electrophoresis of replication intermediates. The long term goal of this research is to understand how triplex-forming DNA repeated sequences affect the regulation of DNA replication in vivo. %%% DNA triplex structures conform a stable barrier to DNA replication under physiological conditions. The long term goal of this research is to understand how triplex-forming repeated DNA sequences affect the regulation of DNA replication in living cells. ***