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:
Katufumi Ohsumi 1
Graduate Students:
Keiko Taki
Hiroko Urawa
Technical Staff:
Kohji Hayashi
Yasushi Takeuchi


Elucidation of dynamic molecular mechanism of replication, recombination and repair processes of genome in prokaryotes and eucaryotes requires extensive research. We focus on interactive processes between replication and recombination. In 1997, the following three interactive subjects were given detailed attention. (1) We analyzed molecular mechanisms of a recombinational hotspot in E. coli, named HotH and we found that collision between replication and rRNA gene transcription is responsible for activation of HotH. (2) We developed a new system, in which the SOS response was induced by replication fork blocking at the Ter site located on pUC derivative plasmids. (3) I (T. H) became head of Japan E. coli genome project in April 1995 and we began to define nucleotide sequences from 13 minutes on the E. coli circular linkage map, in the clockwise direction, together with eleven independent groups of investigators in japan. In January 1997, we reached the region at 69 minutes, sequence data from where to 13 min region being registered in databank. Thus, we expressed the entire E. coli genome in a single continuous nucleotide sequence.


I. Analysis of E. coli recombinational hot spots, Hot.

In E. coli RNase H defective (rnh-) mutants, we identified 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 (HotA-H) of such Hot DNA, 7 of which (HotA-G) were clustered in a narrow region, known as replication terminus region (about 280 kb) located on the circular E. coli genome. Analysis of the HotA, B and C revealed that blocking of the replication fork at the Ter (replication terminus) site is responsible for these Hot activities. Further analysis led to design of a putative model, in which the ds (double stranded)-break occurs at the fork arrested at the DNA replication fork blocking (Ter) site. Through this site the RecBCD recombinational enzyme complex enters the ds-DNA molecule and enhances recombination between directly repeated Hot DNA when the enzyme complex meets an appropriately oriented recombinational hotspot sequence, called Chi.

The Chi sequence had been considered to distribute and orient randomly over the E. coli genome. Dr. Fred Blattner's group in the U. S. A. noted an orientation-dependent symmetrical distribution of the Chi sequence related to replication origin (oriC), at least in a relative narrow region. Our E. coli genome analysis, as will be described below, revealed that this feature extends all over the genome, as shown in Figure 1. This finding is consistent with our model mentioned above; that is if the replication fork is impeded by one cause or another, the ds-break occurred cannot be repaired by recombination until the Chi sequence in an orientation opposite that of replication fork is present at an appropriate site.

Figure 1.
Orientation-dependent distribution of Chi sequence on E. coli genome
Blue and purple bars indicate the number of the Chi site in clock- and counter-clock-wise orientations in a each 100 kb interval of the genome, respectively. The total number of each oriented Chi site is almost the same. However, number of one oriented Chi are apparently dominant over that of the other in one of two half ori-ter sections and vise versa. Black arrow indicates direction of replication fork initiated from replication origin (ori) and an oriented fork is efficiently repaired by recombination using reverse-oriented Chi sequence when it is broken down.

To determine how other termination event-independent Hots can be activated, we selected HotH because it locates outside of replication terminus region. HotH is a 11.2 kb EcoRI DNA fragment and locates at about 91 minutes on the E. coli genetic map. We found that it contains the 3' end region of a rRNA operon, rrnD, and also a Chi sequence at the downstream region of the rrnD. HotH activity was abolished, when a mutation was introduced into the Chi sequence to destroy its activity, which means that the Chi is probably required for HotH activity. To investigate events near the HotH site, we asked whether progress in the DNA replication fork is blocked or retarded in or around of HotH region, and for this we used 2 dimensional (2D) agarose gel electrophoresis. We detected the replication fork passing through this region, in the counterclockwise direction specifically in the rnh- mutant and not in wild type strain. Movement of the counterclockwise directed fork was retarded in a latter part of the rrnD operon, the transcriptional orientation of which is opposite to that of the fork, thereby suggesting that collision between replication fork and rRNA gene transcription may be responsible for retardation. Inactivation of the promoter of the rrnD operon reduced simultaneously retardation of fork movement and HotH activity. Thus, we concluded that HotH activation is caused by collision between DNA replication fork and reverse-oriented rrnD transcription, through the same mechanism related to fork blocking at the Ter site, as described above. This conclusion suggests that the collision is deleterious to the cells, because continuous recombinational repair has to take place to maintain integrity of the replication fork. In Figure 2, because Ter sites are arranged as if they prevent replication fork from colliding with reverse-oriented transcription of rrn operons; circular E. coli genome can be divided into two parts, one is rrn operon side and other is Ter side and they never overlap, which strongly suggests that prevention of collision between fork and transcription may be the physiological role of the Ter site.

Figure 2.
Arrangement of rrn operons and Ter sites on the E. coli genome.
Reds arrows indicate locations and transcriptional direction of rrn operons. means DNA replication terminus (Ter) site which can block replication fork approaching from the right.


II. SOS inducibility by replication fork blocking at Ter site on plasmid and its dependency on distance from ori to Ter sites.

We developed a new system, in which the SOS response was induced by replication fork blocking at the Ter site located on pUC derivative plasmids. In the system, production of Ter binding protein, known as Tus protein, can be controlled by placing the structural gene, tus, under the araC promoter and SOS induction is fully controlled by omitting or adding arabinose. The extent of SOS response was controlled by measuring the activity of b-galactosidase, the expression of which is under control of the sfiA gene, a typical SOS responsive one, the product of which inhibits cell division. SOS inducibility dependent on recA+ and lexA+ genes. Using this system, we found that plasmids at a long distance from plasmid ori to Ter were SOS positive but ones with a short distance were SOS negative. Analyses revealed that up to 2.5 kb, the SOS response is hardly induced, but in a more extended range to at least 4.8 kb, inducibility showed good linear correlation to the distance. Replication intermediate molecules of the SOS+ and SOS- plasmids were purified through CsCl density gradient centrifugation and tertiary structures were analyzed using 2D gel electrophoresis and electron-microscopy. In particular we examined the region of single stranded (ss) DNA found at the Y-junction at the ori or Ter site, because ssDNA functions as a cofactor for in vitro activation of RecA protein; none of differences between them found. While the cause of ori-Ter length dependent SOS induction needs more study, one possibility is that a ds-break occurs at the arrested fork and degradation product from the ds-end may trigger SOS induction. In such case, a longer DNA to be degraded may more efficiently induce the SOS response. Another possibility is that some specific tertiary structure of the q type DNA molecule may be responsible for SOS induction, and to form the structure, a longer ori-Ter DNA may be needed.


III. The E. coli genome project

E. coli genome project in Japan was begun in 1989, the objective being to analyze, in an independently living organism, nucleotide sequences from 0 min in a clockwise direction. In April 1995, DNA sequences of the 0-12.7 min region in the genetic map were determined, published or registered. At that time, I (T. H.) was appointed leader of the Japan E. coli genome project and a group of researchers was re-organized to analyzed DNA sequences of the region from 12.7 min to 70 min, using Kohara lambda clones and the shot-gun sequence technique.

In January 1997, we determined about a 2.2 mega nucleotide sequence of a region corresponding to 12.7-69.0 min (including DNA replication terminus region). Combining these data with data (69.0-100 min) of Dr. Fred Blattner and colleagues in the USA, the entire E. coli genome was expressed in a single, continuous nucleotide sequence, in which about 3400 putative open reading frames were identified. At this time, BlattnerŐs group independently determined the entire E. coli DNA sequence. Figures 1 and 2 show arrangements of Chi sequences and Ter sites on whole genome, respectively; these are some results of the genomic sequence analysis. Our sequence data, registered in the DNA Data Bank in Japan (DDBJ) are also available through Web sites (http://mil.geges.nig.ac.jp/ecoli/, http://www.ddbj.nig.ac.jp/, http://bsw3.aist-nara.ac.jp/).


Publication List:
Yamamoto, Y., Aiba, H., et al. (1997) Construction of a contiguous 874-kb sequence of the Escherichia coli-K12 genome corresponding to 50.0-68.8 min on the linkage map and analysis of its sequence features. DNA research 4, 91-113.

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Last Modified: 12:00, May 28, 1998