INITIATION OF LAGGING-STRAND SYNTHESIS
滞后链合成的启动
基本信息
- 批准号:3285790
- 负责人:
- 金额:$ 27.82万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1984
- 资助国家:美国
- 起止时间:1984-07-01 至 1996-12-31
- 项目状态:已结题
- 来源:
- 关键词:DNA binding protein DNA directed DNA polymerase DNA primase DNA replication DNA replication origin Escherichia coli crosslink electron microscopy enzyme activity enzyme substrate complex gel mobility shift assay gene mutation helicase immunoelectron microscopy intermolecular interaction mutant nucleic acid structure southern blotting temperature sensitive mutant
项目摘要
The replication fork is a complex structure. Upward of 20 different
protomers are likely to operate together in an enzyme machine that has
been termed the replisome. In Escherichia coli, this protein
conglomerate, which is made up of primosomal proteins and the DNA
polymerase III holoenzyme (itself composed of 10 subunits), moves on the
parental DNA at 1000 nt/sec, simultaneously unwinding the template and
synthesizing the nascent leading- and lagging-strands in a coordinated
fashion. It is our goal to understand how these replication proteins
assemble on the DNA, to illuminate the interactions that hold the
replication fork together, and to determine how each protein at the
replication fork contributes to the orderly process of semi-conservative
DNA replication. To do so, we study: i) The independent activities of
individual replication proteins, ii) the nature of various partial
reactions catalyzed by subsets of the complete complement of replication
fork proteins, iii) the control circuits and parameters that affect
Okazaki fragment synthesis catalyzed by replication forks reconstituted
in vitro with purified primosomal proteins and the DNA polymerase III
holoenzyme, and iv) the phenotypes of E. coli mutant strains deficient
in the PriA, PriB, and PriC replication proteins.
Our studies on replication fork action have allowed us to develop a model
describing control of Okazaki fragment synthesis. A key feature of this
model is the interaction between DnaB (the replication fork helicase) and
DnaG (the primase), and between DnaB and DnaC (a primosomal protein).
These aspects of the model will be tested by isolating new mutant
proteins that are defective in their interactions. These will be
examined, along with existing mutant proteins, in the reconstituted
replication fork system for their affect on Okazaki fragment synthesis.
Studies on the action of the subassemblies of the DNA polymerase III
holoenzyme have suggested differential participation of the subunits in:
i) nascent strand synthesis, ii) the interaction between the leading- and
lagging-strand polymerase complex, iii) preinitiation and initiation
complex formation on the new primer terminus on the lagging strand, and
iv) in mediating the disassembly of the lagging-strand polymerase after
termination of Okazaki fragment synthesis and in its transit to the new
primer terminus. The roles of the Pol III HE subunits at the fork will
be defined by reconstituting replication forks with individually purified
subunits.
The precise coordination required between proteins and DNA strands at the
replication fork, and the important role of specific protein-protein
interactions in replication fork action, implies a defined molecular
architecture. We will study the protein-DNA structures present at the
replication fork using replication proteins labelled metabolically to
high specific activity, gel shift analysis, electron microscopy and
immunoelectron microscopy.
The validity of our models on replication fork action will be tested in
vivo by studying Okazaki fragment synthesis in strains where DnaG
production can be controlled. The role of the PriA, PriB, and PriC
primosomal proteins in cellular replication, and the reason that PriA
inactivation induces the SOS response, will be probed by studying Okazaki
fragment synthesis and replication fork movement in priA, priB, and priC
strains.
复制叉是一个复杂的结构。 超过20种不同的
原异构体很可能在一个酶机器中一起运作,
被称为复制体。 在大肠杆菌中,这种蛋白质
聚合体,由原染色体蛋白质和DNA组成
聚合酶III全酶(本身由10个亚基组成),
亲本DNA以1000 nt/sec的速度,同时解旋模板,
以协调的方式合成新生的前导链和滞后链,
时尚. 我们的目标是了解这些复制蛋白
组装在DNA上,以阐明维持DNA的相互作用。
复制叉在一起,并确定每个蛋白质在
复制叉有助于半保守的有序过程
DNA复制。 为了做到这一点,我们研究:一)独立的活动,
单个复制蛋白,ii)各种部分复制蛋白的性质,
由完全复制补体亚群催化的反应
叉蛋白,iii)影响的控制电路和参数
重组复制叉催化的冈崎片段合成
在体外用纯化的primosomal蛋白和DNA聚合酶III
全酶; iv)E.大肠杆菌缺陷突变株
PriA、PriB和PriC复制蛋白。
我们对复制叉行为的研究使我们能够建立一个模型
描述了冈崎片段合成的控制。 其中一个关键特征是,
模型是DnaB(复制叉解旋酶)和
DnaG(引发酶),以及DnaB和DnaC(一种引发体蛋白)之间。
将通过分离新的突变体来测试模型的这些方面
在相互作用中有缺陷的蛋白质。 这些将是
检查,沿着现有的突变蛋白,在重组
复制叉系统对冈崎片段合成影响。
DNA聚合酶Ⅲ酶解作用的研究
全酶已经表明亚基在以下方面的不同参与:
i)新生链合成,ii)前导链和
滞后链聚合酶复合物,iii)预起始和起始
在滞后链上的新引物末端上形成复合物,和
iv)介导滞后链聚合酶的分解,
冈崎片段合成的终止及其向新的
引物末端 Pol III HE亚基在分叉处的作用将
通过用单独纯化的
亚单位。
蛋白质和DNA链之间所需的精确协调,
复制叉的重要作用,以及特异性蛋白质-蛋白质
在复制叉行动中的相互作用,意味着一个定义的分子
架构 我们将研究存在于
使用代谢标记的复制蛋白质的复制叉,
高比活性、凝胶位移分析、电子显微镜和
免疫电镜
我们的模型在复制分叉操作上的有效性将在
体内通过研究冈崎片段合成的菌株,其中DnaG
生产可以控制。 PriA、PriB和PriC的作用
Primosomal蛋白在细胞复制中的作用,以及PriA
失活诱导SOS反应,将通过研究冈崎
priA、priB和priC中的片段合成和复制叉移动
菌株
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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KENNETH J MARIANS其他文献
KENNETH J MARIANS的其他文献
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{{ truncateString('KENNETH J MARIANS', 18)}}的其他基金
Mechanisms of DNA Replication, Chromosome Compaction, and Chromosome Unlinking
DNA 复制、染色体压缩和染色体解联机制
- 批准号:
10618506 - 财政年份:2018
- 资助金额:
$ 27.82万 - 项目类别:
Mechanisms of DNA Replication, Chromosome Compaction, and Chromosome Unlinking
DNA 复制、染色体压缩和染色体解联机制
- 批准号:
9900025 - 财政年份:2018
- 资助金额:
$ 27.82万 - 项目类别:
Mechanisms of DNA Replication, Chromosome Compaction, and Chromosome Unlinking
DNA 复制、染色体压缩和染色体解联机制
- 批准号:
10373984 - 财政年份:2018
- 资助金额:
$ 27.82万 - 项目类别:
Integrated PhD Training Program in Cancer Biology
癌症生物学综合博士培训计划
- 批准号:
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- 资助金额:
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7492914 - 财政年份:2006
- 资助金额:
$ 27.82万 - 项目类别:
Integrated PhD Training Program in Cancer Biology
癌症生物学综合博士培训计划
- 批准号:
7220759 - 财政年份:2006
- 资助金额:
$ 27.82万 - 项目类别:
Integrated PhD Training Program in Cancer Biology
癌症生物学综合博士培训计划
- 批准号:
7669223 - 财政年份:2006
- 资助金额:
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