HUMAN GENOME CLONING AND ISOLATION OF SPECIFIC DNAS IN YEAST

工作总结：内部相关重复元素之间的分离 基因组可能导致相邻序列的改变， 易位我们正在研究的机制和遗传控制， 细菌和模式真核生物中分歧的DNA之间的重组， 酿酒酵母用大肠我们已经解决了 错配修复系统（MMR）如何防止重组的问题， 在杂交DNA形成之前或期间起作用的术语。两 使用不同的DNA在体外制备不同类型的杂交分子， 转化成野生型和各种错配缺陷突变体。两 结果，高质粒存活率和优先损失的尾 链，表明MMR系统通过作用于 链交换步骤，可能是通过切除入侵链。我们 我认为，其他人观察到的非常低的重组频率 研究人员在共轭实验中， DNA是由于优先损失的入侵链， 异源双链体形成。 这项工作的下一阶段是研究 纯化的MutS蛋白与高度错配的异源双链体的结合 体外 此外，这些异源双链体和各种 E.大肠杆菌错配修复系统将检查使用 电镜 利用酵母，我们研究了错配修复系统在 高度发散分子之间的重组（28%）。 这 重组是由特定的DNA组织（反向 重复）和由于DNA突变而改变的复制 聚合酶D聚合结构域。 这两个因素在协同作用 导致住房增加多达1000倍的方式 重组不匹配修复无法防止自修复 重组这些观察是我们以前工作的后续， 提出了一种新的机制，涉及复制的双重- 链断裂诱导分化DNA之间的重组。

Summary of Work: Recombination between related, repeat elements within the genome may lead to alterations in adjacent sequences or translocations. We are investigating mechanisms and genetic control of recombination between diverged DNAs in bacteria and the model eukaryote, the yeast Saccharomyces cerevisiae. With E. coli we have addressed questions of how mismatch repair systems (MMR) prevent recombination in terms of acting prior to or during the formation of hybrid DNAs. Two types of hybrid molecules were prepared in vitro, using diverged DNAs and transformed into wild type and various mismatch deficient mutants. Two results, high plasmid survival and preferential loss of the tailed strand, suggest that the MMR system prevents recombination by acting at the strand exchange step, probably by excision of the invading strand. We propose that the very low recombination frequencies observed by other investigators in conjugation experiments involving similarly diverged DNAs are due to preferential loss of the invading strand during heteroduplex formation. The next phase of this work is to study the binding of purified MutS protein to highly mismatched heteroduplexes in vitro. In addition, complexes between these heteroduplexes and various components of the E. coli mismatch repair system will be examined using electron microscopy. Using yeast we have examined the role of mismatch repair system in recombination between highly diverged (28%) molecules. This recombination was facilitated by specific DNA organization (inverted repeats) and by altered replication due to a mutation in the DNA polymerase d polymerization domain. Both factors acted in a synergistic manner causing together as much as 1000-fold increase of homeologous recombination. Mismatch repair was unable to prevent homeologous recombination. These observations follow up on our previous work where we suggested a novel mechanism for the involvement of replication in double- strand break induced recombination between diverged DNAs.