Homologous recombination and gene amplification induced by DNA replication fork inhibition

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Before starting this project, we had already found that (1) in E. coli, DNA replication fork blocking event at a replication fork terminus (Ter) activates homologous recombination at the nearby sister chromosomal regions, converting these into recombinational hotspots, Hot, and (2) in S. cerevisiae, Fobl protein is required for blocking DNA replication at DNA replication fork barrier site called RFB in each ribosomal RNA gene (rDNA) repeat. From the observation that, in fob1-mutant, both processes, recombination between rDNA and amplification of rDNA, did not occur, replication blocking event at RFB site was required for rDNA amplification as well as rDNA recombination.From the subsequent research in this project, the following have been established ;(1)One of the "Hot" regions, HotA, near the TerB site of the E. coli chromosome was found to be also amplified probably through rolling circle type replication; the degree of the amplification, which occurred in 10 % of the cell population … More , was as high as approximately 400-fold on average.(2)Although it was already known that Sir2, one of the silencing proteins in yeast, is required for suppressing both PolII-dependent expression and recombination within rDNA repeats, the mechanism remained undetermined. We found that under sir2 defective conditions, suppression of specific PolII-dependent bi-directional transcriptions from the promoter E-pro, located between 35S rDNA and 5S rDNA, was abrogated. This results in the dissociation of cohesin from the chromosomal DNA and decreases the association between sister-chromosomes, resulting in the activation of unequal recombination between rDNA repeats and changing the copy number of rDNA.(3)In fobl mutant, the copy number of rDNA is fixed at a high level. In order to elucidate this maintenance mechanism, we collected six mutants in which the rDNA copy number was drastically decreased only under fobl- conditions. We found that all six mutations occurred in condensin subunit genes. Subsequence analysis of localization of condensin in rDNA region revealed that condensin was Fobl-dependently associated with RFB site in S phase, thereby suggesting that condensin is essential for maintenance of a long rDNA tandem array.Shinohara's group tried to identify new protein factors associated with Dmcl, a meiosis-specific yeast, RecA homologue, and found two new proteins, previously called Mei5 and Sae3 proteins. They showed three proteins form a complex and play a role in meiotic specific recombination, because their binding to chromosome in meiosis is mutually dependent. Less