Regulation of DNA double-strand break repair pathway choice
Regulation of DNA double-strand break repair pathway choice
批准号:
10656805
负责人:
Wolf-Dietrich Heyer
金额:
$46.66万
依托单位国家:
美国
项目类别:
财政年份:
2023
资助国家:
美国
项目状态:
未结题
起止时间:
2023-07-01 至 2028-06-30
关键词:
AbbreviationsAffectBRCA2 geneBase PairingBiochemicalBiochemical ReactionC-terminalCamptothecinCell CycleCellsChromosomal RearrangementClinicalComplexCyclin-Dependent KinasesDNA BindingDNA Binding DomainDNA DamageDNA Double Strand BreakDNA Repair GeneDNA biosynthesisDNA damage checkpointDNA lesionDNA replication forkDNA-Directed DNA PolymeraseDataDefectDouble Strand Break RepairDrug TargetingEnergy TransferFilamentFluorescenceFluorescence Resonance Energy TransferGenesGenomeGenome StabilityGenomicsGoalsHumanImageImmune checkpoint inhibitorIn VitroIndividualIonizing radiationKnowledgeLengthMediatingMediatorMitosisModalityModelingMutagenesisNonhomologous DNA End JoiningPathway interactionsPatient SelectionPhasePoly(ADP-ribose) Polymerase InhibitorPoly(ADP-ribose) PolymerasesPolymerasePredispositionProcessProductivityPropertyProteinsProtocols documentationRAD52 geneRad51 recombinaseRadiation induced damageReactionReactive Oxygen SpeciesRegimenRegulationResearchResistanceRoleS phaseSideSourceTestingTherapeuticTopoisomerase IIType I DNA Topoisomerasescancer cellcancer therapychromosome fusionchromosome lossclinical applicationhomologous recombinationinhibitorinnovationinsightloss of functionmutantnovelpersonalized medicineprotein purificationreconstitutionrepairedresponsetranslational potentialtumor
中文摘要
项目摘要
拟议研究的总体目标是确定合成杀伤力的机制基础
同源重组(HR)缺陷和单链退火缺陷之间的网络
(RAD 52)或POLq介导的末端连接(TMEJ)。令人兴奋的初步数据显示,BRCA 2和RAD 52
延迟TMEJ对S期相关DNA双链断裂(DSB)的修复,直至M期。这
调节避免了TMEJ介导的单侧DSB的染色体重排,
复制叉断裂。我们的方法结合了创新的细胞周期相分辨成像的DNA修复
蛋白质和DNA损伤标记物,并使用纯化的人蛋白质进行机械生化分析,
重组反应。我们的研究结果将有潜在的翻译意义的临床应用,
新开发的用于治疗HR缺陷型肿瘤的RAD 52和POLq抑制剂
方案、患者选择和DNA损伤反应检查点抑制剂的使用以及对
聚(ADP-核糖)聚合酶抑制。
具体目标是:
1.明确BRCA 2在DSB修复途径控制中的作用机制。我们将测试模型,
BRCA 2的DNA结合特性对于TMEJ抑制至关重要。在目标1A中,我们进行基础研究
以确定全长BRCA 2的基本DNA结合特性。在目标1B中,我们将定义
BRCA 2的结构域是细胞中TMEJ抑制所必需的。这种基于细胞和生物化学的结合
研究将确定TMEJ抑制所需的BRCA 2的功能和区域。
2.明确BRCA 2和RAD 52抑制TMEJ的机制。BRCA 2和RAD 52采用两种不同的
抑制POLq的DNA聚合酶活性的模式,这可能影响TMEJ中的其他反应步骤
过程我们将在体外用纯化的蛋白质重建TMEJ,以确定其机制,
BRCA 2(Aim 2A)和RAD 52(Aim 2B)抑制TMEJ。我们将测试总体TMEJ反应的抑制,
单个步骤包括1)DNA结合,2)末端比对和3)DNA合成。分析野生型和
将在体外和细胞中进行POLq的催化突变。
3.定义哪些HR缺陷易受RAD 52功能丧失的影响。这是一个悬而未决的问题,
RAD 52的表达将导致POLq介导的染色体融合和所有HR缺陷背景中的致死性(目的
3A)或所有BRCA 2突变体(Aim 3B)。我们的初步研究表明,BRCA 2的负载是一个模型,
这是将TMEJ限制在M期的关键步骤,并且经过该步骤的HR缺陷不受RAD 52抑制的影响。
英文摘要
Project Summary
The general goal of the proposed research is to define the mechanistic underpinning for the synthetic lethality
network between a deficiency in homologous recombination (HR) and defects in single-strand annealing
(RAD52) or POLq-mediated end-joining (TMEJ). Exciting preliminary data revealed that BRCA2 and RAD52
delay the repair of S-phase-associated DNA double-stranded breaks (DSB) by TMEJ until M-phase. This
regulation avoids TMEJ-mediated chromosomal rearrangements of one-sided DSBs that are produced by
replication fork breakage. Our approach combines innovative cell cycle phase resolved imaging of DNA repair
proteins and DNA damage markers with mechanistic biochemical analysis using purified human proteins in
reconstituted reactions. Our results will have potential translational implications for the clinical application of
newly developed RAD52 and POLq inhibitors for the treatment of HR-deficient tumors with respect to application
protocols, patient selection, and use of DNA damage response checkpoint inhibitors as well as the response to
poly(ADP-ribose) polymerase inhibition.
The Specific Aims are:
1. Define the mechanism of action of BRCA2 in DSB repair pathway control. We will test the model that the
DNA binding properties of BRCA2 are critical for TMEJ inhibition. In Aim 1A, we conduct foundational studies
to determine the fundamental DNA binding properties of full-length BRCA2. In Aim 1B, we will define which
domains of BRCA2 are required for TMEJ inhibition in cells. This combination of cell-based and biochemical
studies will define the functions and regions of BRCA2 that are required for TMEJ inhibition.
2. Define the mechanism of TMEJ inhibition by BRCA2 and RAD52. BRCA2 and RAD52 employ two different
modes to inhibit the DNA polymerase activity of POLq which may affect additional reaction steps in the TMEJ
process. We will reconstitute TMEJ in vitro with purified proteins to determine the mechanisms by which
BRCA2 (Aim 2A) and RAD52 (Aim 2B) inhibit TMEJ. We will test inhibition of the overall TMEJ reaction and
individual steps including 1) DNA binding, 2) end-alignment, and 3) DNA synthesis. Analysis of wild type and
catalytic mutants of POLq will be conducted in vitro and in cells.
3. Define which HR defects are susceptible to RAD52 loss of function. It is an open question whether loss
of RAD52 will lead to POLq-mediated chromosome fusions and lethality in all HR-deficient backgrounds (Aim
3A) or all BRCA2 mutants (Aim 3B). Our preliminary studies suggest a model that loading of BRCA2 is the
critical step to limit TMEJ to M-phase and that HR defects past this step are not affected by RAD52 inhibition.
期刊论文(0)
专著(0)
科研奖励(0)
会议论文
Genome instability induced by homologous recombination
-
批准号:10019104
-
项目类别:
-
资助金额:$31.4万
-
财政年份:2020
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
Genome instability induced by homologous recombination
-
批准号:10437930
-
项目类别:
-
资助金额:$31.4万
-
财政年份:2020
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
Genome instability induced by homologous recombination
-
批准号:10795314
-
项目类别:
-
资助金额:$1.92万
-
财政年份:2020
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
Genome instability induced by homologous recombination
-
批准号:10241424
-
项目类别:
-
资助金额:$31.4万
-
财政年份:2020
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
Genome instability induced by homologous recombination
-
批准号:10670239
-
项目类别:
-
资助金额:$31.4万
-
财政年份:2020
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
Genome instability induced by homologous recombination
-
批准号:10806624
-
项目类别:
-
资助金额:$1.13万
-
财政年份:2020
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
2015 Nucleic Acids Gordon Research Conference
-
批准号:8892554
-
项目类别:
-
资助金额:$0.55万
-
财政年份:2015
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
FASEB SRC: Genetic Recombination and Genome Rearrangements
-
批准号:8525768
-
项目类别:
-
资助金额:$0.3万
-
财政年份:2013
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
Functions of Rad51 Paralogs in Recombinational DNA Repair
-
批准号:8519965
-
项目类别:
-
资助金额:$48.87万
-
财政年份:2011
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
Functions of Rad51 Paralogs in Recombinational DNA Repair
-
批准号:8890651
-
项目类别:
-
资助金额:$53.03万
-
财政年份:2011
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
Functions of Rad51 Paralogs in Recombinational DNA Repair
-
批准号:8703631
-
项目类别:
-
资助金额:$51.44万
-
财政年份:2011
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
Functions of Rad51 Paralogs in Recombinational DNA Repair
-
批准号:8338889
-
项目类别:
-
资助金额:$51.99万
-
财政年份:2011
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
Functions of Rad51 Paralogs in Recombinational DNA Repair
-
批准号:8237897
-
项目类别:
-
资助金额:$51.83万
-
财政年份:2011
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
DNA DAMAGE CHECKPOINT MEDIATED REGULATION OF DOUBLE-STRAND BREAK REPAIR
-
批准号:8171262
-
项目类别:
-
资助金额:$0.08万
-
财政年份:2010
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
RECOMBINATIONAL MECHANISMS OF DNA REPAIR IN EUKARYOTES
-
批准号:8171388
-
项目类别:
-
资助金额:$0.24万
-
财政年份:2010
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
DNA DAMAGE CHECKPOINT MEDIATED REGULATION OF DOUBLE-STRAND BREAK REPAIR
-
批准号:7957786
-
项目类别:
-
资助金额:$0.33万
-
财政年份:2009
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
RECOMBINATIONAL MECHANISMS OF DNA REPAIR IN EUKARYOTES
-
批准号:7957775
-
项目类别:
-
资助金额:$0.33万
-
财政年份:2009
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
RECOMBINATIONAL MECHANISMS OF DNA REPAIR IN EUKARYOTES
-
批准号:7723651
-
项目类别:
-
资助金额:$0.81万
-
财政年份:2008
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
Microbial Production Core Facility
-
批准号:7211306
-
项目类别:
-
资助金额:$22.64万
-
财政年份:2007
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
DNA DAMAGE CHECKPOINT MEDIATED REGULATION OF DOUBLE-STRAND BREAK REPAIR
-
批准号:7602190
-
项目类别:
-
资助金额:$0.08万
-
财政年份:2007
-
负责人:Wolf-Dietrich Heyer
-
依托单位:
海外基金