NATIONAL INSTITUTE FOR BASIC BIOLOGY
DIVISION OF GENE EXPRESSION REGULATION II
- Professor:
- 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.
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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;
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- (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.
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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.
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- 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.
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- 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.
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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.