NATIONAL INSITUTE FOR BASIC BIOLOGY

National Institute for Basic Biology

DIVISION OF GENE EXPRESSION AND REGULATION II

Professor:
Takashi Horiuchi
Research Associates:
Masumi Hidaka
Takehiko Kobayashi
Ken-ichi Kodama
Katsuki Johzuka
Post Doctoral Fellow:
Katsufumi Ohsumi
Keiko Taki
Graduate Students:
Hiroko Urawa
Technical Staff:
Kohji Hayashi
Yasushi Takeuchi


Homologous recombination which may occur in all organisms apparently involves exchange between two parent-derived chromatids plus the repair of DNA damage induced by physical and chemical reagents. As deduced from our analyses of recombination hotspots of E. coli and S. cerevisiae, in particular the activity related to DNA replication fork blocking events, the physiological function of homologous recombination, especially in normally growing cells is better understood. In 1998, work on the two following interrelated subjects have advanced our knowledge of related factors.


I. Molecular mechanism of expansion/contraction of ribosomal RNA gene repeats in S. cerevisiae.

HOT1 is a mitotic recombinational hotspot in the yeast S. cerevisiae and was first identified by Keil and Roeder (1984). HOT1 stimulates both intra- and inter-chromosomal recombination nearby regions when inserted at a non-rDNA location. HOT1 was originally cloned on a 4.6 kb BglII-A fragment which locates in rRNA repeated genes (about 150 copies) on chromosome XII. A single rRNA unit consists of two transcribed 35S and 5S rRNA genes and two non-transcribed regions, NTS1 and NTS2, the former is between 3'-ends of 35S and 5S rRNA genes, and the latter is 5' ends of these two genes. The HOT1 DNA fragment contains the NTS1, 5SRNA and NTS2 region but it was later found to be composed of two non-contiguous cis-elements, E and I, located in NTS1 and NTS2, respectively. Because E and I positionally and functionally overlapped with the enhancer and initiator of the 35S rRNA transcription, respectively, Roeder's group suggested that transcription by RNA polymerase I, initiated at the 35S rRNA promoter site may stimulate recombination of the downstream region, thereby revealing Hot1 activity.

The NTS1 has a site at which the replication fork is blocked and we termed this site SOG, but later called RFB (replication fork barrier). By assaying activity for various DNA fragments derived from the NTS1 and cloned on plasmids, we determined the minimal region, about 100 bp long, located near or within the enhancer region of the 35S rRNA transcription and essential for blocking replication fork advancing in a direction opposite that for transcription. The RFB sequence has no homology to any other known sequence and has no characteristic structure such 2-fold symmetry, repeated structure, etc.; hence, trans-factor(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.

To investigate functional relationships between the fork blocking activity in RFB and the hotspot activity in HOT1, we first isolated mutants defective in Hot1 activity and examined whether these mutations would also affect RFB activity. Using a colony color sectoring assay method, we isolated 23 Hot defective mutants from approximately 40,000 mutagenized colonies. Among these Hot- mutants, one proved to be a rad52 mutant; the other 4 mutants lose fork blocking activity. Genetic analysis of these mutants revealed that all four were recessive for the RFB phenotype and defined one complementation group. This mutation was designated fob1 (fork blocking function) and one of the fob1 mutants, fob1-4, was further analyzed. First, from yeast cDNA bank, we cloned FOB1 gene by selecting a DNA fragment, which had suppressive activity for Hot deficiency of the mutant. The minimal FOB1 plasmid was shown to complement both the Hot- and RFB- phenotype of the fob1-4 mutant, suggesting that both phenotypes are caused by a mutation in the FOB1 gene. DNA sequencing of the FOB1 gene revealed that the putative Fob1 protein consists of 566 amino acids and has a molecular mass of 65,000 daltons. This gene has no homology with any DNA sequence registered in Genbank. Sequencing of the fob1-4 mutant gene revealed two mutational changes in the open reading frame, one is non-sense (amber) and other is a miss-sense mutation. The amber mutation may account for the two defective phenotypes of the fob1-4 mutant and why it is non-leakyness.

Because there is an extraordinary functional similarity between factors required for the hotspot activity in E. coli and S. cerevisiae, in both organisms ds-break and repair would occur when the DNA replication fork is arrested at fork blocking sites and probably was spontaneously blocked.

Next we investigated relationship between FOB1 gene and expansion/contraction of rRNA gene (rDNA) repeats in collaboration with the laboratory of Dr. Masayasu Nomura in Univ. of California (Irvine). As described above, S. cerevisiae carries approximately 150 copies of rRNA gene in tandem repeats. .It was found that the absence of an essential subunit of RNA polymerase I (PolI) in rpa135 deletion mutants triggers a gradual decrease in rDNA repeat number to about one-half the normal level. Reintroduction of the missing RPA135 gene induced a gradual increase in repeat number back to the normal level . Gene FOB1 was shown to be essential for both the decrease and increase of rDNA repeats (Fig.1). Thus, DNA replication fork blockage appears to stimulate recombination and play an essential role in rDNA expansion/contraction and sequence homogenization, and possibly, in the instability of repeated sequences in general (ex. expansion of triplet repeats). RNA polymerase I, on the other hand, appears to control repeat numbers, perhaps by stabilizing rDNA with the normal repeat numbers as a stable nucleolar structure.

Figure 1.
Increase of the size of chromosome by Fob1 and PolI proteins. (by Pulse-field gel electrophoresis)

We propose a model, shown in Fig.2, we call the fork block dependent recombination model to show how the blocked replication fork changes the copy number of rDNA.

Figure 2.
Fork block-dependent recombination model for rDNA expansion/contraction.


II. The E. coli genome project

E. coli genome project in Japan was started in 1989, the objective being to analyze, in an independently living organism, nucleotide sequences from 0 min in a clockwise direction. In April 1995, the DNA sequences of the 0-12.7 min region in the genetic map were determined and they were published or registered. At that time I (T. H.) was appointed leader of the Japan E. coli genome project and a new group of researchers was reorganized to analyze DNA sequences of the region from 12.7 min to 70 min of W3110 strain, using Kohara lambda clones and a new protocol, including a shot-gun sequence technique.
In January 1997, we had completed a sequence analysis of E. coli genome by combining our sequence data of 2.2 mega base of a region corresponding to 12.7-69.0 min (including DNA replication terminus region) with those (69.0-100 min) previously registered in GenBank by other groups. The entire E. coli genome was expressed in a single, continuous nucleotide sequence (4,636,552 bp), in which about 4.300 putative open reading frames were identified. At this same time, Blattneršs group in Wisconsin Univ. had determined independently the sequence of entire genome of another closely related strain, MG1655. Now we are sequencing the entire genome of W3110 (it will be completed within this year) and their genomic structural changes between W3110 and MG1655 strains, which occurred during a very short period after being separated from a common ancestor, are being compared. It may reveal about mechanisms of micro-evolution in bacteria.


Publication List:
Kobayashi, T., Rein, T., & Depamphilis, ML. (1998) Identification of primary initiation sites for DNA replication in the hamster dihydrofolate reductase gene initiation zone. Mol Cell Biol 18, 3266-77
Kobayashi, T., Heck, J.D., Nomura, M., and Horiuchi, T. (1998) Expansion and contraction of ribosomal DNA repeats in Saccharomyces cerevisiae: requirement of replication fork blocking (Fob1) protein and the role of RNA polymerase I. Genes & Development 12, 3821-3830.

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Last Modified: 12:00, May 28, 1999