Assembly and Activation of Enzyme-ssDNA Complexes
Assembly and Activation of Enzyme-ssDNA Complexes
批准号:
8214651
负责人:
SCOTT W MORRICAL
金额:
$32.08万
依托单位国家:
美国
项目类别:
财政年份:
1993
资助国家:
美国
项目状态:
已结题
起止时间:
1993-08-01 至 2015-01-31
关键词:
ATP HydrolysisAntibioticsBacteriophage T4BindingBiochemicalBiochemistryCellsCommunicable DiseasesComplexDNADNA biosynthesisDNA replication forkDefectDiagnosisEnzyme ActivationEnzymesExhibitsFilamentFluorescenceGenetic RecombinationGenomeKineticsLightLinkMaintenanceMalignant NeoplasmsMediator of activation proteinMethodsModelingMolecular ConformationMutagenesisOrganismPathway interactionsPolymerasePreventionProcessPropertyProteinsRecruitment ActivityResearchSS DNA BPSedimentation processSingle-Stranded DNASpectrum AnalysisSystemTestingThermodynamicsantitumor drugcancer therapycrosslinkfluorescence imaginghelicasehuman diseasepresynapticprogramsprotein protein interactionrecombinaserecombinational repairsingle moleculetreatment strategytumor
中文摘要
这项研究计划的目的是确定重组酶
在噬菌体中,解旋酶被组装到单链DNA(SsDNA)上
T4DNA复制/重组系统。我们将研究T4 UvsX的组装
重组到突触前细丝,我们将研究两种不同DNA的组装
解旋酶,gp41和dda,在复制分叉和重组中间产物上的单链DNA上。
这三种酶都必须在已经被紧密覆盖的细胞中组装到单链DNA上。
结合Gp32,T4单链DNA结合蛋白。UvsX和gp41都需要
特异性介体蛋白,分别为UvsY或GP59,用于正确组装到Gp32-ssDNA上
复合体,而DDA通过蛋白质-蛋白质直接相互作用达到同样的效果
使用Gp32。我们将使用经典生化来探索所有这三种酶的负载机制。
方法(动力学、热力学、荧光法、沉淀法、交联法)、
分子方法(荧光成像、力光谱)和诱变。我们的
具体目标是:(1)确定UvsX-ssDNA突触前的动力学机制
细丝组装和坍塌。我们将测试一个模型,在该模型中,UvsY蛋白选择性地
增强单丝成核,UvsX主动取代单链DNA中的gp32,单丝表现出
与三磷酸腺苷水解有关的动态不稳定性。(2)确定T4GP59蛋白的相互作用
通过复制分叉,DNA控制解旋酶组装和聚合酶阻断。我们将测试一个
Gp32与滞后链单链DNA的协同结合模型将GP59从
聚合酶阻断到解旋酶装载构象,它将gp41解旋酶招募到
复制分叉。(3)确定与Gp32的相互作用如何调节DNA解旋酶
T4Dda蛋白的功能。我们将测试一个模型,在该模型中dda-Gp32蛋白-蛋白质
相互作用促进DDA齐聚并增强其DNA解离性能
复制和重组事务。了解解旋酶和
重组酶正确组装到单链DNA上是理解
DNA复制、重组和修复机制,在所有生物体中都是保守的。
DNA复制/重组/修复机制中的错误与
包括癌症在内的人类疾病状态。了解重组酶和解旋酶是如何
因此,正确组装和激活单链DNA复合体可有助于预防,
癌症的诊断和治疗。
英文摘要
The objective of this research program is to determine the mechanisms by which recombinase
and helicase enzymes are assembled onto single-stranded DNA (ssDNA) in the bacteriophage
T4 DNA replication/recombination system. We will study the assembly of the T4 UvsX
recombinase into presynaptic filaments, and we will study the assembly of two different DNA
helicases, Gp41 and Dda, onto ssDNA at replication forks and in recombination intermediates.
All three enzymes must assemble onto ssDNA in the cell that is already covered with tightly
bound Gp32, the T4 ssDNA-binding protein. UvsX and Gp41 both require the activity of a
specific mediator protein, UvsY or Gp59, respectively, for proper assembly onto Gp32-ssDNA
complexes, whereas Dda achieves the same effect through direct protein-protein interactions
with Gp32. We will explore all three enzyme loading mechanisms using classical biochemical
methods (kinetics, thermodynamics, fluorescence, sedimentation, crosslinking), single-
molecule approaches (fluorescence imaging, force spectroscopy), and mutagenesis. Our
SPECIFIC AIMS are: (1) Determine the kinetic mechanism of UvsX-ssDNA presynaptic
filament assembly and collapse. We will test a model in which UvsY protein selectively
enhances filament nucleation, UvsX actively displaces gp32 from ssDNA, and filaments exhibit
dynamic instability linked to ATP hydrolysis. (2) Determine how interactions of T4 Gp59 protein
with replication fork DNA control helicase assembly and polymerase blockage. We will test a
model in which cooperative binding of Gp32 to lagging-strand ssDNA converts Gp59 from a
polymerase-blocking to a helicase-loading conformation that recruits Gp41 helicase to the
replication fork. (3) Determine how interactions with Gp32 modulate the DNA helicase
functions of T4 Dda protein. We will test a model in which Dda-Gp32 protein-protein
interactions promote the oligomerization of Dda and enhance its DNA unwinding properties in
both replication and recombination transactions. Understanding how helicase and
recombinase enzymes are correctly assembled onto ssDNA is fundamental to understanding
DNA replication, recombination, and repair mechanisms that are conserved in all organisms.
There are clear links between errors in DNA replication/recombination/repair machineries and
human disease states including cancer. Understanding how recombinase- and helicase-
ssDNA complexes are correctly assembled and activated may therefore aid in the prevention,
diagnosis, and treatment of cancer.
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Kinetics of presynaptic filament assembly in the presence of single-stranded DNA binding protein and recombination mediator protein.
单链 DNA 结合蛋白和重组介体蛋白存在下突触前丝组装的动力学。
DOI:
10.1021/bi401060p
发表时间:
2013
期刊:
Biochemistry
影响因子:
2.9
作者:
[Liu,Jie, Berger,ChristopherL, Morrical,ScottW]
通讯作者:
Morrical,ScottW
Coordination of DNA replication and recombination activities in the maintenance of genome stability.
DOI:
10.1002/jcb.23211
发表时间:
2011-10
期刊:
JOURNAL OF CELLULAR BIOCHEMISTRY
影响因子:
4
作者:
[Maher, Robyn L., Branagan, Amy M., Morrical, Scott W.]
通讯作者:
Morrical, Scott W.
The gene 59 protein of bacteriophage T4 modulates the intrinsic and single-stranded DNA-stimulated ATPase activities of gene 41 protein, the T4 replicative DNA helicase.
噬菌体 T4 的基因 59 蛋白调节基因 41 蛋白(T4 复制 DNA 解旋酶)的内在单链 DNA 刺激的 ATP 酶活性。
DOI:
--
发表时间:
1994
期刊:
The Journal of biological chemistry
影响因子:
--
作者:
[Morrical,SW, Hempstead,K, Morrical,MD]
通讯作者:
Morrical,MD
Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY.
重组介体蛋白 UvsY 对 T4 基因 32 蛋白 DNA 结合活性的调节。
DOI:
10.1016/j.jmb.2008.05.039
发表时间:
2008
期刊:
Journal of molecular biology
影响因子:
5.6
作者:
[Pant,Kiran, Shokri,Leila, Karpel,RichardL, Morrical,ScottW, Williams,MarkC]
通讯作者:
Williams,MarkC
Helicase assembly protein Gp59 of bacteriophage T4: fluorescence anisotropy and sedimentation studies of complexes formed with derivatives of Gp32, the phage ssDNA binding protein.
噬菌体 T4 的解旋酶组装蛋白 Gp59:与噬菌体 ssDNA 结合蛋白 Gp32 衍生物形成的复合物的荧光各向异性和沉降研究。
DOI:
10.1021/bi010116n
发表时间:
2001
期刊:
Biochemistry
影响因子:
2.9
作者:
[Xu,H, Wang,Y, Bleuit,JS, Morrical,SW]
通讯作者:
Morrical,SW
共 10 条
STRUCTURE AND FUNCTION OF HOMOLOGOUS RECOMBINATION ENZYMES
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批准号:6997980
-
项目类别:
-
资助金额:$19.55万
-
财政年份:2004
-
负责人:SCOTT W MORRICAL
-
依托单位:
Homology Directed Repair
-
批准号:8327274
-
项目类别:
-
资助金额:$19.64万
-
财政年份:2004
-
负责人:SCOTT W MORRICAL
-
依托单位:
Homology Directed Repair
-
批准号:8381905
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项目类别:
-
资助金额:$24.27万
-
财政年份:2004
-
负责人:SCOTT W MORRICAL
-
依托单位:
Homology Directed Repair
-
批准号:8725060
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项目类别:
-
资助金额:$23.76万
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财政年份:2004
-
负责人:SCOTT W MORRICAL
-
依托单位:
Homology Directed Repair
-
批准号:8543550
-
项目类别:
-
资助金额:$32.56万
-
财政年份:2004
-
负责人:SCOTT W MORRICAL
-
依托单位:
Homology Directed Repair
-
批准号:7992616
-
项目类别:
-
资助金额:$20.25万
-
财政年份:2004
-
负责人:SCOTT W MORRICAL
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依托单位:
ASSEMBLY AND ACTIVATION OF ENZYME SSDNA COMPLEXES
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批准号:2396916
-
项目类别:
-
资助金额:$3.09万
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财政年份:1996
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负责人:SCOTT W MORRICAL
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依托单位:
Assembly and Activation of Enzyme-ssDNA Complexes
-
批准号:6544460
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项目类别:
-
资助金额:$33.41万
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财政年份:1993
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负责人:SCOTT W MORRICAL
-
依托单位:
Assembly and Activation of Enzyme-ssDNA Complexes
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批准号:6920725
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项目类别:
-
资助金额:$30.22万
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财政年份:1993
-
负责人:SCOTT W MORRICAL
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依托单位:
Assembly and Activation of Enzyme-ssDNA Complexes
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批准号:8018659
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项目类别:
-
资助金额:$31.61万
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财政年份:1993
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负责人:SCOTT W MORRICAL
-
依托单位:
ASSEMBLY AND ACTIVATION OF ENZYME-SSDNA COMPLEXES
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批准号:2691547
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项目类别:
-
资助金额:$23.08万
-
财政年份:1993
-
负责人:SCOTT W MORRICAL
-
依托单位:
ASSEMBLY AND ACTIVATION OF ENZYME-SSDNA COMPLEXES
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批准号:6018942
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项目类别:
-
资助金额:$27.28万
-
财政年份:1993
-
负责人:SCOTT W MORRICAL
-
依托单位:
Assembly and Activation of Enzyme-ssDNA Complexes
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批准号:6779881
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项目类别:
-
资助金额:$31.82万
-
财政年份:1993
-
负责人:SCOTT W MORRICAL
-
依托单位:
ASSEMBLY AND ACTIVATION OF ENZYME-SSDNA COMPLEXES
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批准号:2186351
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项目类别:
-
资助金额:$17.77万
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财政年份:1993
-
负责人:SCOTT W MORRICAL
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依托单位:
ASSEMBLY AND ACTIVATION OF ENZYME/SSDNA COMPLEXES
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批准号:2910824
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项目类别:
-
资助金额:$2.33万
-
财政年份:1993
-
负责人:SCOTT W MORRICAL
-
依托单位:
ASSEMBLY AND ACTIVATION OF ENZYME-SSDNA COMPLEXES
-
批准号:3308307
-
项目类别:
-
资助金额:$16.21万
-
财政年份:1993
-
负责人:SCOTT W MORRICAL
-
依托单位:
ASSEMBLY AND ACTIVATION OF ENZYME-SSDNA COMPLEXES
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批准号:6385793
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项目类别:
-
资助金额:$27.01万
-
财政年份:1993
-
负责人:SCOTT W MORRICAL
-
依托单位:
Assembly and Activation of Enzyme-ssDNA Complexes
-
批准号:7464795
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项目类别:
-
资助金额:$32.5万
-
财政年份:1993
-
负责人:SCOTT W MORRICAL
-
依托单位:
Assembly and Activation of Enzyme-ssDNA Complexes
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批准号:7786972
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项目类别:
-
资助金额:$32.41万
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财政年份:1993
-
负责人:SCOTT W MORRICAL
-
依托单位:
ASSEMBLY AND ACTIVATION OF ENZYME-SSDNA COMPLEXES
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批准号:2186349
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项目类别:
-
资助金额:$16.36万
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财政年份:1993
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负责人:SCOTT W MORRICAL
-
依托单位:
海外基金