Studies on replication fork blocking system and its physiological function in yeast.

复制叉阻断位点（SOG）位于rRNA重复基因中（约140个拷贝）。单个rRNA单元由两个转录的35 S和5S rRNA基因以及两个非转录区域NTS 1和NTS 2组成。NTS 1有一个复制分叉被阻止的站点。通过测定SOG活性的各种DNA片段来自NTS 1和克隆的质粒，我们确定了最小的区域，约100 bp长，位于附近的增强子区域的35 S rRNA转录，该区域包含在E元件，两个顺式元件之一，酵母重组热点HOT 1活性必不可少的。我们还发现，它是足够的叉阻断复制前进的方向相反，35 S rRNA转录。SOG序列与任何其他已知序列没有同源性，并且没有特征性结构，例如2重对称性、重复结构等;因此，反式因子可能在阻断叉中起作用。为了研究SOG和HOT 1活性之间的功能关系，分离HOT 1缺陷突变体，并使用2D琼脂糖凝胶电泳检测其叉阻断活性。在HOT 1阴性突变体中，我们发现了一个在同源重组中包含的基因缺陷的rad 52突变体。我们还发现了另一种类型的突变体同时缺陷HOT 1和SOG活动。多效性突变体的分离和分析表明，复制叉阻断事件可能需要在酵母中增强同源重组。类似的增强机制可能在其他真核生物和原核生物中起作用。

Replication fork blocking site, we termed this site SOG,is located in rRNA repeated genes (about 140 copies). A single rRNA unit consists of two transcribed 35S and 5S rRNA genes and two non-transcribed regions, NTS1 and NTS2. The NTS1 has a site at which the replication fork is blocked. By assaying SOG activity for various DNA fragments derived from the NTS1 and cloned on plasmids, we determined the minimal region, about 100 bp long, located near the enhancer region of the 35S rRNA transcription ; this region is contained in the E element, one of two cis-elements essential for yeast recombinational hotspot HOT1 activity. We also found that it is adequate for fork blocking replication advancing in a direction opposite that for 35S rRNA transcription. The SOG sequence has no homology to any other known sequence and has no characteristic structure such as 2-fold symmetry, repeated structure, etc. ; hence, a trans-factor (s) may have a role in blocking the fork.To examine the functional relationship between SOG and HOT1 activities, HOT1 defective mutants were isolated and their fork blocking activities were examined using 2D agarose gel electrophoresis. Among HOT1 negative mutants, we found a rad52 mutant defective in a gene included in homologous recombination. We also found another type of mutant simultaneously defective in HOT1 and SOG activities. Isolation and analysis of the pleiotropic mutants suggested that replication fork blocking events may be required to enhance homologous recombination in yeast. A similar enhancing mechanism may function in other eucaryotes and prokaryotes.