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

复制分叉阻断位点，我们称之为SOG，位于rRNA重复基因(约140个拷贝)中。单个rRNA单位由两个转录的35S和5S rRNA基因和两个非转录区域NTS1和NTS2组成。NTS1有一个复制分叉被阻止的站点。通过分析来自NTS1的各种DNA片段的SOG活性，我们确定了位于35S rRNA转录增强子区附近的最小区域，约100bp，该区域包含在E元件中，E元件是酵母重组热点HOT1活性所必需的两个顺式元件之一。我们还发现，它足以阻止复制以与35S rRNA转录相反的方向进行。SOG序列与其他已知序列没有同源性，也没有2重对称性、重复结构等特征结构，因此，反式因子(S)可能具有阻断分叉的作用。在HOT1阴性突变体中，我们发现一个rad52突变体在同源重组中的一个基因上存在缺陷。我们还发现另一种类型的突变体同时在HOT1和SOG活性上存在缺陷。多效性突变体的分离和分析表明，复制分叉阻断事件可能需要增强酵母中的同源重组。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.