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A. DEPARTMENT OF
MOLECULAR GENETICS
A-d. Division of Nucleic Acid Chemistry - Tsutomu
Katayama Group
RESEARCH
ACTIVITIES
(1)
Molecular mechanism of DNA replication-coupled
inactivation of the initiator protein in
Escherichia coli: Interaction of DnaA with
the sliding clamp-loaded DNA and the sliding
clamp-Hda complex
Masayuki Su'etsugu, Makoto Takata1,
Toshio Kubota2, Yusaku Matsuda and
Tsutomu Katayama (Present addresses:
1Sumitomo Pharmaceuticals Co., Osaka,
Japan; 2Department of Pharmacy, Kyushu
University Hospital, Fukuoka, Japan)
--In Escherichia
coli, the ATP-DnaA protein initiates
chromosomal replication. After the DNA polymerase
III holoenzyme is loaded onto DNA, DnaA-bound ATP
is hydrolyzed in a manner depending on Hda protein
and the DNA-loaded form of the DNA polymerase III
sliding clamp subunit, which yields ADP-DnaA, an
inactivated form for initiation. This regulatory
DnaA-inactivation represses extra initiation
events. In this study, in vitro replication
intermediates and structured DNA mimicking
replicational intermediates were first used to
identify structural prerequisites in the process of
DnaA-ATP hydrolysis. Unlike duplex DNA loaded with
sliding clamps, primer RNA-DNA heteroduplexes
loaded with clamps were not associated with
DnaA-ATP hydrolysis, and duplex DNA provided in
trans did not rescue this defect. At least
40-bp duplex DNA is competent for the DnaA-ATP
hydrolysis when a single clamp was loaded. The
DnaA-ATP hydrolysis was inhibited when ATP-DnaA was
tightly bound to a DnaA box-bearing
oligonucleotide. These results imply that the
DnaA-ATP hydrolysis involves the direct interaction
of ATP-DnaA with duplex DNA flanking the sliding
clamp. Furthermore, Hda protein formed a stable
complex with the sliding clamp. Based on these, we
suggest a mechanical basis in the DnaA-inactivation
that ATP-DnaA interacts with the Hda-clamp complex
with the aid of DNA binding.4)
(2)
DiaA, a novel DnaA-binding protein, ensures the
timely initiation of E.coli chromosome
replication
Takuma Ishida, Nobuyoshi Akimitsu1,
Tamami Kashioka2, Masakazu
Hatano3, Toshio Kubota4,
Yasuyuki Ogata5, Kazuhisa
Sekimizu1 and Tsutomu Katayama (Present
addresses: 1Graduate School of
Pharmaceutical Sciences, University of Tokyo,
Japan; 2Institute for Chinese Medicine,
Nakamura City Hospital, Kochi, Japan;
3Santen Pharmaceutical Co., Nara, Japan;
4Department of Pharmacy, Kyushu
University Hospital, Fukuoka, Japan;
5Radioisotope Center, National Institute
of Genetics, Mishima, Shizuoka, Japan)
--The DnaA protein is
the initiator of E. coli chromosomal
replication. In this study, we identify a novel
DnaA-associating protein, DiaA, which is required
for the timely initiation of replication during the
cell cycle. DiaA promotes the growth of specific
temperature-sensitive dnaA mutants and
ensures stable minichromosome maintenance, while
DiaA does not decrease the cellular DnaA content. A
diaA::Tn5 mutation suppresses the
cold-sensitive growth of an overinitiation-type
dnaA mutant independently of SeqA, a
negative modulator of initiation. Flow cytometry
analyses reveal that the timing of replication
initiation is disrupted in the diaA mutant
cells as well as wild-type cells with pBR322
expressing the diaA gene. Gel-filtration and
chemical cross-linking experiments show that
purified DiaA forms a stable homodimer.
Immunoblotting analysis indicates that a single
cell contains about 280 DiaA dimers. DiaA
stimulates minichromosome replication in an in
vitro system especially when the level of DnaA
included is limited. Moreover, specific and direct
binding between DnaA and DiaA is observed, which
requires a DnaA N-terminal region. DiaA binds to
both ATP- and ADP-bound forms of DnaA with a
similar affinity. Thus, we conclude that DiaA is a
novel DnaA-associating factor that is crucial to
ensure the timely initiation of chromosomal
replication.2)
(3)
Reactivation of DnaA by DNA sequence-specific
nucleotide exchange in vitro
Kazuyuki Fujimitsu and Tsutomu Katayama
--In Escherichia
coli, ATP-bound DnaA protein can initiate
chromosomal replication. After initiation, DnaA-ATP
is hydrolyzed by interactions with a complex
containing a replicase subunit to yield the
inactive ADP-DnaA. However, the mechanisms which
regenerate ATP-DnaA from ADP-DnaA are not well
understood. We report here that a 70-bp DNA segment
promotes exchange of the DnaA-bound nucleotide in a
sequence specific manner, thus reactivating the
initiation function of DnaA in vitro. This
segment contains a typical DnaA-binding 9-mer
motif, the DnaA box, and two DnaA box-like
sequences. The presence and precise composition of
these three motifs are required for the
DnaA-reactivating activity, which suggests that a
highly ordered complex which includes multimeric
DnaA molecules is formed for isomerization of DnaA.
We named this DNA segment DARS, for
DnaA-reactivating sequence. The role of DARS in
regulation of DnaA function in vivo is
discussed.1)
(4)
Novel heat shock protein HspQ stimulates the
degradation of mutant DnaA protein in
Escherichia coli
Toh-ru Shimuta, Kiyotaka Nakano1,
Yoko Yamaguchi, Shogo Ozaki, Kazuyuki Fujimitsu,
Chika Matsunaga, Kenji Noguchi2, Akiko
Emoto3 and Tsutomu Katayama (Present
addresses: 1Chugai Pharmaceutical Co.,
Tokyo, Japan; 2Kaketsuken Institute,
Kumamoto, Japan; 3Department of
Pharmacy, Saga University Hospital, Saga,
Japan)
--Escherichia
coli DnaA protein initiates chromosomal
replication and is an important regulatory target
during the replication cycle. In this study, a
suppressor mutation isolated by transposon
mutagenesis was found to allow growth of the
temperature-sensitive dnaA508 and
dnaA167 mutants at 40℃. The suppressor
consists of a transposon insertion in a previously
annotated ORF, here termed hspQ, a novel
heat shock gene whose promoter is recognized by the
major heat shock sigma factor σ32.
Expression of hspQ on a pBR322 derivative
inhibits growth of the dnaA508 and
dnaA167 mutants at 30℃, whereas growth of
dnaA46 and other dnaA mutants is
insensitive to changes in the level of hspQ.
Cellular DnaA508 protein is degraded rapidly at
elevated temperature, but hspQ disruption
impedes this process. In contrast, DnaA46 protein
is rapidly degraded in an hspQ-independent
manner. Gel-filtration and chemical cross-linking
experiments suggest that HspQ forms a stable
homodimer in solution and can form homomultimers
consisting of about four monomers. Heat
shock-induced proteases such as Clp contain
homomultimers of subunit. We propose that HspQ is a
new factor involved in the quality control of
proteins and that it functions by excluding
denatured proteins.3)
PUBLICATIONS
Papers
1. Fujimitsu, K. and Katayama, T. (2004).
Reactivation of DnaA by DNA sequence-specific
nucleotide exchange in vitro. Biochem.
Biophys. Res. Commun., 322, 411-419.
2. Ishida, T., Akimitsu, N., Kashioka, T., Hatano,
M., Kubota, T., Ogata, Y., Sekimizu, K. and
Katayama, T. (2004). DiaA, a novel DnaA-binding
protein, ensures the initiation timing of
Escherichia coli chromosome replication. J.
Biol. Chem., 279, 45546-45555.
3. Shimuta, T., Nakano, K., Yamaguchi, Y., Ozaki,
S., Fujimitsu, K., Matsunaga, C., Noguchi, K.,
Emoto, A. and Katayama, T. (2004). Novel heat-shock
protein HspQ stimulates the degradation of mutant
DnaA protein in Escherichia coli. Genes
Cells, 9, 1151-1166.
4. Su'etsugu, M., Takata, M., Kubota, T., Matsuda,
Y. and Katayama, T. (2004). Molecular mechanism of
DNA replication-coupled inactivation of the
initiator protein in Escherichia coli:
Interaction of DnaA with the sliding clamp-loaded
DNA and the sliding clamp-Hda complex. Genes Cells,
9, 509-522.
Reviews
5. 片山
勉(2004)｢大腸菌染色体複製の開始とその制御に関わるAAA+スーパーファミリータンパク質｣生化学76,
pp.1440-1443.
Books
6. 片山
勉,末次正幸,川上広宣(2004)｢大腸菌ゲノムDNA複製系｣,ゲノミクスとプロテオミクスの新展開〜生物情報の解析と応用〜(エヌ･ティー･エス,今中忠行監修)pp.57-64.
ORAL
PRESENTATIONS
1. Katayama, T. DnaA protein in Escherichia
coli: Regulation and functional structure for
initiation of chromosomal DNA replication. The 1st
Asian Symposium for Pharmaceutical Sciences,
Fukuoka, Japan, January, 2004.
2. Katayama, T. DNA replication-dependent feedback
against DnaA initiator activity in E. coli.
Keystone Symposia on Bacterial Chromosomes, Santa
Fe, NM, USA, February, 2004. (Invited speaker)
3. Katayama, T. Mechanism of DnaA-ATP hydrolysis
that depends on the sliding clamp of the replicase
in E. coli. Approach to molecular mechanisms
of DNA transactions from archaeal research,
Fukuoka, Japan, April, 2004.
4. 片山
勉「大腸菌染色体の複製開始制御におけるDNAポリメラーゼの役割」生体防御P&P/難治性疾患リサーチコア合同研究会、福岡、2004年5月
5. 藤光和之、片山
勉「特異的DNA配列による染色体複製開始蛋白質DnaAの活性化」21世紀大腸菌研究会―蛋白質と遺伝子の細胞内動態の共役:モデル生物大腸菌を用いた統合の試み―、沼津、2004年6月
6. 末次正幸、紫牟田透、片山
勉「Hda-スライディングクランプ複合体によるDnaA活性制御機構の解析」第17回DNA複製・分配ワークショップ、仙台、2004年7月
7. 川上広宣、片山
勉「大腸菌複製開始蛋白DnaAにおけるAAA+アルギニンフィンガーモチーフの役割」第77回日本生化学会大会ワークショップ、横浜、2004年10月
8. 毛谷村賢司、末次正幸、植田 正、片山
勉「大腸菌DnaAのスライディングクランプ依存的不活性化の分子機構:DnaAのDNA結合ドメインの解析」日本遺伝学会第76回大会、大阪、2004年9月
9.
尾崎省吾、藤光和之、毛谷村賢司、川上広宣、末次正幸、胡桃坂仁志、片山
勉「超好熱性真性細菌Thermotoga
maritima由来DnaA蛋白質におけるDNA複製開始能の解析」日本遺伝学会第76回大会、大阪、2004年9月
POSTER
PRESENTATIONS
1. 松田雄作、末次正幸、植田 正、片山
勉「大腸菌スライディングクランプ結合因子の網羅的探索」第17回DNA複製・分配ワークショップ、仙台、2004年7月
2. 石田琢磨、片山
勉「大腸菌染色体の複製開始タイミング制御に必要な新規因子DiaAはDnaAと直接結合する」第27回日本分子生物学会年会、神戸、2004年12月
3. 紫牟田透、末次正幸、片山
勉「Hdaタンパク質の機能構造解析:スライディングクランプとの相互作用およびDnaA結合性ATP加水分解に関する部位の同定」第27回日本分子生物学会年会、神戸、2004年12月
4. 山口陽子、末次正幸、藤光和之、石田琢磨、片山
勉「大腸菌染色体複製制御因子hda遺伝子の低温感受性変異株の分離と解析」第27回日本分子生物学会年会、神戸、2004年12月
5. 古郡麻子、樋口久美子、片山 勉、日高真澄、堀内
嵩、岩井成憲、真木寿治「複製フォーク進行阻害回避における損傷乗り越えDNAポリメラーゼの役割:oriCプラスミドin
vitro
DNA複製系を用いた解析」第27回日本分子生物学会年会、神戸、2004年12月
SOCIAL ACTIVITIES AND
OTHERS
1. Dr. Katayama was awarded a Junior
Research Leader of Kyushu University.
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