DNA Triplexes and Replication

[9723924] Mirkin本研究将重点关注DNA三联体在DNA复制中的作用。在之前的研究中，我们发现在体外DNA聚合之前或过程中形成的DNA三联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模板中的三联体区域，而完全能够解开相应的双联体区域。因此，在生理条件下，DNA三联体可以形成DNA聚合酶的稳定屏障。我们假设，DNA三联体也可能抑制DNA在体内的复制。为了支持这一假设，我们最近发现一个特殊的三联体形成DNA序列d(G)n(d(C)n，在大肠杆菌细胞中阻断复制叉的运动。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。这些结果非常提示在体内由d(G)n(d(C)n形成的一种不寻常的结构可能是导致复制停止的原因。本研究的具体目的是获得d(G)n(d(C)n重复序列的三重结构导致复制停止的直接证据，并将这些研究扩展到包括不同的三重结构形成重复。我们将完成d(G)n(d(C)n重复的详细突变分析，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，我们将使用结构特异性化学物质氯乙醛对大肠杆菌细胞中含有(G)n(d(C)n的质粒进行化学探测。我们还将利用复制中间体的双向电泳分析不同长度和组成的三联体重复序列对大肠杆菌细胞质粒DNA复制的影响。本研究的长期目标是了解三联体形成的DNA重复序列如何影响体内DNA复制的调节。%%% DNA三重结构在生理条件下是DNA复制的稳定屏障。这项研究的长期目标是了解三联体形成的重复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. ***