National Insitute for Basic Biology

DIVISION OF GENE EXPRESSION AND REGULATION II

Professor:
Takashi Horiuchi
Research Associate:
Masumi Hidaka
Takehiko Kobayashi
Ken-ichi Kodama
Institute Research Fellow:
Katsuki Johzuka
Graduate Student:
Katsufumi Ohsumi
Keiko Taki
Technical Staff:
Yasushi Takeuchi
Homologous recombination may occur in all organisms. While related functions apparently involve exchange between two parent-derived chromatids and repair of DNA damage incurred by physical and chemical reagents, many questions remain unanswered. As deduced from our analyses of recombinational hotspots of E. coli & 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.

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

We identified hotspots of recombination (termed Hot) on the E. coli chromosome. These sites are specifically activated under rnh- (RNase H-defective) conditions (Nishitani et al., MGG 240, 307, 1993). Analysis of these Hot 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 (called Ter) site, through which the RecBCD recombinational enzyme enters the ds-DNA molecule and enhances recombination between directly repeated Hot DNA, when the enzyme meets an appropriately oriented Chi sequence.

If the ds-break is induced by replication fork arrest, physiological effects on the cell would be evident. To elucidate effects of fork arrest at Ter sites, we constructed an E. coli strain, termed a blocked strain, in which oligonucleotides with the TerA sequence were inserted into the lacZ gene in an orientation so as to block the fork of clockwise (normal direction) replication. In this strain, replication forks were expected to be blocked at both the TerL in the lacZ and at TerA, D and E sites. While the blocked strain grew somewhat more slowly than a control strain, it had abnormal phenotypes similar of E. coli dam- mutants, i.e., hyper-Rec phenotype, recA +- and recB + (C +)-dependent growth, and constitutive SOS expression. We propose that the following sequential events may occur in both strains (see Figure 1). A ds-break occurs at the blocked replication fork in the blocked strain and at the ongoing fork in the dam - mutant, through which RecBCD enzyme enters and degrades the dsDNA molecule, and the degradation product serves as the signal molecule for SOS induction. When RecBCD enzyme meets an appropriately oriented Chi sequence, its DNase activity is converted to recombinase enzyme, which is able to form a new replication fork, recombinationally with RecA and SSB proteins. This model (i) explains the puzzling phenotype of recA - and recB - (C -) mutants and SOS-inducing phenotype of polA, lig and dna mutants under restrictive conditions, (ii) provides an interpretation for the role of the Chi sequence, and (iii) suggests a possible key role for homologous recombination with regard to cell survival following the arrest of DNA replication (Horiuchi and Fujimura; J Bacteriol. in press).

Figure 1

Fig. 1.
Model of rescue from blocked DNA replication in E. coli, as applicable to a spontaneously stalled replication fork. Essentially the same sequential reactions would probably occur in S. cerevisiae.

II. Analysis of recombinational hot spot in S. cerevisiae.

HOT1 is a mitotic recombinational hotspot in the yeast S. cerevisiae and was first identified by Keil and Roeder. 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 4.6kb BglII fragment which locates in rRNA repeated genes (about 140 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 gene and NTS2 region but it was later found to be composed of two non-contiguous cis-elements, E and I, Iocated 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. 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 replication fork advancing in a direction opposite that for transcription. The SOG 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, Hotl, activity.

To investigate functional relationships between the fork blocking activity in SOG and the hotspot activity in HOT1, we first isolated mutants defective in Hotl activity and examined whether these mutations would also affect Sog 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 Sog 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 FOB gene by selecting a DNA fragment which had suppressive activity for Hot deficiency of the mutant. The minimal FOG plasmid was shown to complement both the Hot- and Sog- 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 fobl-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 dsbreak and repair would occur when the DNA replication fork is arrested at fork blocking sites and probably was spontaneously blocked (Figure 1 ).

Publication List:

Horiuchi; T., Fujimura, Y., Nishitani, T., Kobayashi, T. and Hidaka, M. (1994) The DNA replication fork blocked at the Ter site may be entrance for the RecBCD enzyme into duplex DNA. J Bacteriol. 176, 4656-4663.