|Budget Amount *help
¥6,300,000 (Direct Cost : ¥6,300,000)
Fiscal Year 1993 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 1992 : ¥4,800,000 (Direct Cost : ¥4,800,000)
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.