Direct analysis of fork blockage and DNA repair in vivo

描述：（由申请人提供）：一些实验室已经推进了我们的 了解如何通过构建合成DNA来处理DNA损伤 含有位点特异性/化学特异性损伤的分子， 将这些分子转化成细胞。受损分子的能力 以产生有活力的后代或特定的突变体后代。而 这些方法提供了许多有用的知识，但它们是有限的，因为 仅在多轮繁殖后分析子代DNA。的 本提案中描述的实验旨在允许直接 在引入后含有位点特异性损伤的DNA的物理分析 活的细菌细胞该方法利用噬菌体X的特定突变体， 它们的头部很小，只有大约15 kb的DNA。这是小号的 足以允许构建含有位点特异性损伤的分子。 含有放射性残留物以便于分析的受损DNA将被 在体外包装成小头部，并组装成噬菌体颗粒。的 颗粒将被纯化，然后用于感染细胞。将准备DNA 从受感染的细胞作为一个函数的时间，以遵循损害的命运。 修复的损害将遵循动力学使用的恢复， 在损伤位置的限制性酶切位点。另外还有按 我实验室的主要兴趣是确定复制分叉是否被阻塞 用二维凝胶电泳检测损伤。实验 这里描述的将尝试使用DNA验证这个系统， 脱碱基位点，这是非常重要的自发和诱导DNA 损害我们希望能够测量脱碱基位点修复的动力学， 野生型和突变株，并且还确定无碱基位点是否阻断 复制分叉。如果该系统得到验证，许多其他类型的DNA损伤 可以在未来的研究中进行分析。

DESCRIPTION: (provided by applicant): A number of labs have advanced our understanding of how DNA damage is processed by constructing synthetic DNA molecules that contain site-specific/chemically specific damage and transforming these molecules into cells. The ability of the damaged molecules to generate viable progeny or particular mutant progeny is then analyzed. While these approaches have provided much useful knowledge, they are limited because the progeny DNA is analyzed only after many rounds of propagation. The experiments described in this proposal are designed to allow the direct physical analysis of DNA containing site-specific damage upon introduction into living bacterial cells. The approach utilizes a specific mutant of phage X that makes small heads, which package only about 15 kb of DNA. This is a size small enough to permit construction of molecules containing site-specific damage. Damaged DNA, containing radioactive residues for ease of analysis, will be packaged in vitro into the small heads, and assembled into phage particles. The particles will be purified and then used to infect cells. DNA will be prepared from the infected cells as a function of time to follow the fate of the damage. Repair of the damage will be followed kinetically using restoration of a restriction enzyme cleavage site at the location of the damage. In addition, a major interest of my lab is to determine whether replication forks are blocked at the damage using 2-dimensional gel electrophoresis. The experiments described here will attempt to validate this system using DNA with and without abasic sites, which are very important in both spontaneous and induced DNA damage. We hope to be able to measure the kinetics of abasic site repair in wild type and mutant strains, and also to determine whether abasic sites block replication forks. If the system is validated, many other kinds of DNA damage can presumably be analyzed in-future studies.