National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION of GENOME DYNAMICS †Professor

HORIUCHI, Takashi

Assistant Professors:
Technical Staff:
Graduate Students:
Technical Assistants:

JOHZUKA, Katsuki
WATANABE, Taka-aki
MOROOKA, Naoki
OKAMOTO, Haruko
ITAZU, Masako
ISHINE, Naomi
YONEZAWA, Harumi
MATSUZAKI, Yoko

Gene amplification provides host organisms with anabundance of gene products. One example is the high copy
number of rDNA (rRNA gene), which enables a large amountof protein production. On the other hand, drug-resistancegenes can become amplified increasing their effectiveness.
These two forms of gene amplification produce products thatdiffer radically in structure. The former is a direct repeat
product and the latter is an inverted repeat product. We
previously clarified the mechanism of the former type ofamplification and here have shed light on the latter.
We have carried out an amplification experiment using
yeast on the assumption that double rolling circle replication(DRCR) is a core reaction in gene amplification. We induced
DRCR by BIR (break induced replication) and obtained twogene amplification products, HSR (homogeneous stainingregion) and DMs (double minutes) (Watanabe and Horiuchi,
EMBO J. 2005). To further confirm this result, we carried out

an experiment in which DRCR was induced by two entirely
different methods, the Cre-lox system and homologous
recombination. We obtained the gene products HSR andDMs in either case respectively. Furthermore, using Chinese
Hamster Ovary (CHO) cells, we induced DRCR by the Crelox system and obtained two gene products. In addition,
scattered products, as are frequently observed in cultured
cells, are obtained. Finally, we found that inversion occurred
frequently in the inverted repeat structure of the products andis dependent on the DRCR process itself.

I. Construction of a new gene amplification
system via DRCR (double rolling circle
replication) using Cre-lox site-specificrecombination

Previously, we developed a gene amplification system in S.
cerevisiae that is based on DRCR, utilizing break-induced
replication (BIR) (Watanabe and Horiuchi, EMBO J. 2005).
This system produced two amplification products resembling
the HSR and DMs of higher eukaryotes. If DRCR is anactual gene amplification mechanism in yeast, a quite
different initiation reaction, which can induce DRCR, should
produce amplification products resembling HSR and DMs.
Thus, we tried to construct a new DRCR amplification
system that is induced by another process, Cre-lox sitespecific recombination. We first predicted that, if Cre
recombination occurs between the two lox sites, one present
on the replicated and the other on the un-replicated region, as
shown in Figure 1 (a), the replication fork should switch thetemplate from the parental (un-replicated) to the sister–
chromatid (replicated) DNA strands. Furthermore, aFigure 1. Cre-lox can initiate template switching, DRCR and CR (convergent replication).

†: This laboratory was closed on 31 March, 2011.

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Figure 2. Three gene amplification products in CHO cells. (top left)
control cell, (top right) HSR type, (bottom left) DMs type, (bottom right)
Scattered type.

combination of the recombination processes, as shown inFigure 1 (b) and (c), could efficiently induce gene
amplification through DRCR or CR (convergent replication).
In fact, this system produced two kinds of gene product:
highly amplified (>100 copies) chromosome HSR products
and acentric multi-copy extra-chromosomal DMs products.
The structures of these products resemble HSRs and DMs ofhigher eukaryotes, respectively. From previous and presentresults, we concluded that DRCR is indeed an amplification
mechanism in budding yeast and can be naturally initiated ifsome structural requirements are satisfied.
Next, we constructed a similar amplification system in
CHO cells, based on DRCR. This system also produced
intra- and extra-chromosomal amplification products
resembling HSRs and DMs (Figure 2. top right, bottom left).
The amplified regions of HSR-type products undergo
intensive rearrangement seen in mammalian gene
amplification. Furthermore, the CHO system produces
scattered-type amplification seen in cancer cells. This system
can serve as a model for amplification of oncogene and drugresistance genes, and may improve the productivity and easeof use of amplification systems that are widely used formaking pharmaceutical proteins in mammalian cells(manuscript submitted for publication).

II. Mechanism of oncogene-type amplification
under natural conditions

Site-specific recombination consists of two elements, ashort specific sequence (cis-element) and a specific protein(trans-element) which recognizes the specific sequence andrecombines efficiently between them. On the other hand,

general recombination consists of a long nonspecific sequence and several sets ofrecombination proteins (Rec proteins), which
recognize homologous sequences and recombineb e t w e e n t h e m . H o w e v e r, h o m o l o g o u srecombination can take the place of site-specificrecombination by replacing a short specificsequence with a long non-specific sequence.
This indicates that if a lox sequence is replaced
by a long sequence, homologous recombination
can induce DRCR. Replacement of the sequencealone should initiate DRCR by itself, in theabsence of any site-specific recombination
protein factors. We confirmed this expectation as
follows: we created a →←→← structure, called
FAIR (Four Alternate Inverted Repeat) at theright end of chromosome VI and anamplification selective marker, leu2d, inserted
within the FAIR structure. This yeast strain wasplated on agar without leucine, Leu+ clones grewout and their chromosomal structure analyzed.
As expected, two types of amplification wereobserved, HSR- and DM -type, both with theexpected repeated structure. The implications ofthese results are very important, namely, that if
FAIR structures form under natural conditions, gene
amplification will occur. In fact, there are studies in which
FAIR structures were observed at an initial stage of drugresistance gene amplification. Furthermore, there is datasuggesting that the BFB cycle can be an initial step ofamplification. From these previous and our present results,
all steps of oncogene-type gene amplification can be deduced
as follows: a double strand break or recombinational
template switching spontaneously occurs on a chromosome,and the BFB cycle initiates. As a result, a di-centricchromosome is produced and ds-breakage occurs again. The
chromosome structure after two cycles of BFB is a perfect
FAIR structure. Thus, this can initiate DRCR and gene
amplification starts.
Using budding yeast, we here demonstrate that the FAIR
structure has the potential to induce DRCR. Thus, we believethat the basic mechanism of oncogene-type gene
amplification has been determined. However, gene
amplification may also be induced by the BFB cycle.
Especially in higher eukaryotes, there are a large number oftransposable elements, among which there should be FAIR
structures. Thus, it is not surprising that DRCR initiates
without any double-stranded breaks. It is very interesting toask the question whether or not this type of gene
amplification does indeed occur (manuscript in preparation).

III. Double rolling circle replication (DRCR) isrecombinogenic.

Homologous recombination plays a critical role inmaintaining genetic diversity as well as genome stability.
Interesting examples showing hyper-recombination are foundin nature. In chloroplast DNA (cpDNA) and the herpes
simplex virus (HSV) genome, DNA sequences flanked byinverted repeats undergo inversion very frequently,

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National Institute for Basic Biology

Evolutionary Biology and Biodiversity

Figure 3. An “only daughter chromatid” model
A. circular-type DRCR, B. Linear-type DRCR

suggesting hyper-recombinational events. However, themechanisms responsible for these events remain unclear. We
previously observed very frequent inversion in a designed
amplification system based on double rolling circle
replication (DRCR). Utilizing the yeast 2μ plasmid, thegenome of which is known to be replicated by DRCR and thegene amplification system in yeast, we demonstrate that
DRCR is closely related to hyper-recombinational events.
Inverted repeats or direct repeats inserted into these systems
frequently caused inversion or deletion/duplication,respectively, in a DRCR-dependent manner. These results
suggest that DRCR is involved in chromosome
rearrangement associated with gene amplification and thereplication of cpDNA and HSV genomes. We propose amodel (Figure 3), termed the “only daughter chromatid” (redlines) model, in which DRCR inevitably produces only
daughter chromatids and is markedly activatedrecombinationally (manuscript in press).

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