Takashi Horiuchi

Research Associates:
Masumi Hidaka
Takehiko Kobayashi

Graduate Student:
Katufumi Ohsumi

Technical Staff:
Yasushi Takeuchi

Homologous recombination on the chromosome is often uniform, however, in both procaryotes and eucaryotes, there are specific regions or sites, named "hotspots" , where homologous recombination occurs at a higher rate. DNA replication origin in procaryotes (phage) is one example. Another example is the "HOT1" site in yeast which has activity to stimulate recombination, homologously, in adjacent regions. As molecular mechanisms involved in enhancing homologous recombination are not fully characterized, related studies, in an attempt at elucidation, are ongoing in our laboratory. Special focus has been directed to termination processes, and homologous recombinations.

I. Analysis of E. coli recombinational hotspots, Hot.

In E. coli, there are replication fork blocking event-dependent recombinational hotspots. In E. coli RNase H defective (rnh-) mutants, we found specific DNA fragments, termed Hot DNA, when DNA in the ccc form is integrated into the E. coli genome by homologous recombination to form a directly repeated structure, a strikingly enhanced excisional recombination between the repeats occurs. We obtained 8 groups of such Hot DNA, 7 of which were clustered in a narrow region, called replication terminus region (about 280 kb) on the circular E. coli genome. A terminus site ( Ter) can impede the replication fork in a polar fashion. The six Ter sites are located approximately symmetrically in terminus and its surrounding region. To block the fork at the Ter site, another protein factor, Ter binding protein encoded in the tau (or tus) gene is required. In tau cells, Hot activity of HotA, B and C DNAs disappears, thereby indicating that the Hot activity is fork arrest-dependent. In rnh- cells, an alternative new replication origin(s) other than an ordinary replication origin (oriC), Iocated at the terminus region is activated so that a newly initiated fork is immediately blocked at one of the Ter sites and consequently, there is an accumulation of stalled forks. It seems fairly certain that the rnh- specific accumulation of the fork (which we confirmed) is the cause of the Hot activity at the nearby site. In addition, at least for HotA activity, the presence of a Chi, an E. coli recombinational hotspot sequence, properly oriented on HotA DNA itself or somewhere between HotA and Ter site, is required. We prepared a putative model (Figure 1), in which the following events may occur;
(a) Chromosomal structure of a HotA DNA transformant, in which repeated HotA DNAs flanks a Kmr fragment. (b) When the DNA replication fork proceeds from left to right(this can occur under rnh- conditions), there is an efficient block against the fork at the TerB site and the resulting Y-shaped molecules accumulate most in the rnh- strain, less in wild type and not at all in the tau strain. (c) A ds-break is introduced, probably by nicking at a single stranded DNA complementary to the newly synthesized lagging strand. (d) A Chi responsible enzyme, RecBCD, enters the duplex DNA through the ds-break, and travels to the Chi site with concomitant degradation of the newly synthesized ds-DNA molecules, by exonucleolytic activity. (e) The Chi sequence modulates the exonucleolytic activity. (f) The resulting enzyme stimulates excisional homologous recombination between repeated HotA DNAs, resulting in production of the ccc Hot-Kmr DNA molecule.

II. Analysis of a yeast recombinational hotspot, HOT1.

In yeast, Saccharomyces cerevisiae, there are also DNA replication fork blocking sites in rRNA repeated genes (about 140 copies) on chromosome XII. A single repeat unit consists of two transcribed 35S and 5S rRNA genes and two non-transcribed regions, NTS1 and NTS2 (Figure 2). The 35S rRNA gene is transcribed by RNA polymerase I, a polymerase specific for 35S rRNA transcription, and transcription of the 5S RNA gene, the direction of which is opposite that of the 35S rRNA, is carried out by RNA polymerase III, another polymerase specific for 5S rRNA and tRNA production. The NTS1 has a site, termed SOG, at which the replication fork is blocked. We obtained evidence that fork blocking activity at the SOG site, termed SOG activity, is expressed not only on the genome but also on the plasmid, suggesting that the SOG site functions in any context (Kobayashi et al., 1992). 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 and essential for blocking the replication fork advancing in a direction opposite that for transcription. The SOG sequence is unique and has no characteristic structure, such as 2-fold symmetry, repeated structure etc., hence, a transfactor(s) may have a role in blocking the fork. Interestingly, this region is included in one of two cis-elements required for a recombinational hotspot, HOT1, activity.

HOT1 is a yeast mitotic homologous recombinational hotspot, identified by Keil and Roeder (1984). HOT1 stimulates both intra- and inter-chromosomal recombination, and for a precise analysis, enhancement of excisional recombination between directly repeated DNAs at its nearby site was investigated. HOT1 was originally cloned on a 4.6 kb BglII fragment (Fig. 2) and it was later found to be composed of two non-contiguous cis-elements, E and I, located in NTS I and NTS2, respectively. Because E and I positionally and functionally overlapped the enhancer and initiator of the 35S rRNA transcription, respectively, they suggested that transcription by RNA polymerase I, initiated at the 35S rRNA promoter site may stimulate recombination of the downstream region, thereby reveal HOT1 activity. However, our finding that this E region contains the SOG site may give another interpretation.

To examine the functional relationship between SOG and HOT1 activities, HOT1 defective mutants were isolated and their fork blocking activities were subjected to 2D agarose gel electrophoresis. One was rad52 mutant defective in a gene included in homologous recombination. The remaining HOT1 mutants are being examined to determined whether or not their fork blocking activity is active.

Publication List:

Nishitani, H., Hidaka, M. and Horiuchi, T. (1993) Specific chromosomal sites enhancing homologous recombination in Escherichia coli mutants defective in RNase H. Mot Gen. Gener 240, 307-314.