| Project/Area Number |
11680682
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Molecular biology
|
|---|
| Research Institution | KYOTO UNIVERSITY (2000)
Kumamoto University (1999) |
|---|
Principal Investigator |
YAMAZOE Mitsuyoshi Kyoto Univ. Graduate school of Med.(Assistant Professor), 医学研究科, 助手 (00284745)
|
|---|
| Project Period (FY) |
1999 – 2000
|
| Project Status |
Completed (Fiscal Year 2000)
|
| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2000: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1999: ¥2,500,000 (Direct Cost: ¥2,500,000)
|
|---|
| Keywords | Escherichia coli / chromosome partitioning / MukFEB protein complex / SeqA Protein / DNA methylation / Smc蛋白 |
|---|
| Research Abstract |
1. Identification and purification of the MukFEB protein complex.
MukF and MukE bind the C-terminal globular domain of MukB.Purified MukFEB complex binds both double-stranded and single-stranded DNA with similar affinity, regardless of methylation state of DNA.An ATPase activity of the MukFEB complex is not affected by the presence of DNA.We have also observed morphology of MukB and the MukFEB complex using electron microscopy. The globular domains of the MukFEB complex are bigger than those of MukB.Moreover the MukFEB complex sometimes forms network structure, connecting each other by globular domain of MukB.
2. Subcellular localization of the SeqA protein.
SeqA binds preferentially to newly replicated hemimethylated DNA and is localized as discrete foci in E.coli cells. We have examined migration of the SeqA clusters precisely using synchronized culture. After initiation of DNA replication, a single SeqA focus at midcell separates into two foci and these two foci abruptly migrate in opposite directions midway through replication from oriC to the terminus. OriC is replicated at midcell but replicated oriC copies remain linked with each other for substantial period.
3. Subcellular localization of the MukB protein.
MukB is localized as discrete foci within nucleoids dependent on the presence of both MukF and MukE.Most newborn cells have two foci of MukB at 1/4 and 3/4 positions in the cell length, while one focus of SeqA at midcell. Subsequently the single SeqA focus divides into two foci and migrate towards each MukB focus.
4. Sequential binding of nascent chromosomal DNA segments with SeqA clusters
To identify DNA segments associated with SeqA molecules in vivo. we preformed chromatin immunoprecipitation assay with anti-SeqA antibody. Using a synchronous culture of chromosomal replication, we have found that SeqA binds to chromosomal segments sequentially from oriC-proximal to oriC-distal position.
|
