Novel pathways that regulate DNA double-strand break repair events in mammalian cells
Novel pathways that regulate DNA double-strand break repair events in mammalian cells
批准号:
10360432
负责人:
Jessica K Tyler
金额:
$42.38万
依托单位国家:
美国
项目类别:
财政年份:
2021
资助国家:
美国
项目状态:
未结题
起止时间:
2021-03-01 至 2026-01-31
关键词:
AddressAgingBiochemistryBiological AssayCRISPR/Cas technologyCellsChromatinChromatin StructureDNADNA Double Strand BreakDNA RepairDNA Repair PathwayDNA biosynthesisDNA lesionDataDouble Strand Break RepairEventExcisionG1 PhaseGene ExpressionGenetic DiseasesGenetic ScreeningGenetic TranscriptionGenomeGenome StabilityGenomicsGoalsGuide RNAHistonesHuman GeneticsHuman bodyLibrariesLongevityMaintenanceMammalian CellMediatingMolecular GeneticsNonhomologous DNA End JoiningPathway interactionsPositioning AttributeProcessProteinsResearchRoleSaccharomycetalesSingle-Stranded DNAVisionbaseds-DNAgenome integritygenome-widehomologous recombinationinnovationnon-histone proteinnovelpreventprogramsrepairedscreeningstructural biologytissue culture
中文摘要
摘要
我们研究的总体愿景是发现组蛋白和非组蛋白相互作用的新机制。
DNA上的蛋白质,即染色质,调节基因组过程和衰老。特别是,我们努力整合
不同的领域,如染色质在基因组稳定性中的作用和染色质在衰老中的作用。使用
结合生物化学,结构生物学,分子遗传学在芽殖酵母,组织培养和
通过全基因组的方法,我们发现染色质在非细胞周期中被分解和重新组装,
不仅基因表达和DNA复制,而且在DNA双链断裂修复期间。我们有
揭示了这些事件的机制基础及其对这些基因组过程的关键影响。更
近年来,我们已经扩展了我们解决的问题,超越了染色质-例如发现
衰老的新机制基础和发现延长寿命的新方法。同样,受我们
最近发现染色质结构减少了DNA双链断裂到单链的过程
DNA(称为DNA末端切除),我们设计了创新的CRISPR/Cas9 gRNA文库筛选
方法,以确定新的活动,调节DNA双链修复过程中的DNA末端切除。
人体内的大多数细胞处于G 0/G1期,过量的DNA末端切除至关重要,
并不发生在这些细胞中。如果发生这种情况,它将阻止DNA修复的唯一途径,
修复G 0/G1期细胞中的DNA双链断裂,即非同源末端连接(NHEJ),
将导致随后同源介导修复的易位和缺失。事实上,
DNA末端切除是选择使用NHEJ或同源
重组(HR)途径修复DNA双链断裂。我们建议,必须建立机制,
在G 0/G1期细胞中进行有限过量DNA末端切除以防止HR,但仍能使足够的DNA
末端加工不可连接的DNA末端以允许NHEJ介导的修复。蛋白质和途径,
调节G 0/G1期细胞中DNA末端切除的程度目前尚不清楚。因此,
该项目旨在发现调控G 0/G1期DNA末端切除的机制
细胞基于我们的专业知识、新颖的基因筛选方法和
令人信服的初步数据。
基因组维持的另一个关键但知之甚少的方面是基因表达是如何影响
在DNA损伤附近“关闭”,以防止转录和DNA修复之间的碰撞
机械.类似地,在DNA双链断裂修复后重新启动转录是至关重要的,但是
机制不明。我们最近发现了一些蛋白质参与使用我们的新的分析
和基因筛选,所以这个计划的第二个主要目标是发现
转录关闭和DNA双链断裂周围的重新启动。
英文摘要
Summary Abstract
The overall vision for our research is to discover novel mechanisms by which histone and non-histone
proteins on DNA, i.e. chromatin, regulate genomic processes and aging. In particular, we strive to integrate
different fields, such as the role of chromatin in genome stability and the role of chromatin in aging. Using a
combination of biochemistry, structural biology, molecular genetics in budding yeast, tissue culture and
genome-wide approaches, we have discovered that chromatin is disassembled and reassembled during not
only gene expression and DNA replication but also during DNA double-strand break repair. We have
revealed the mechanistic bases for these events and their key impact on these genomic processes. In more
recent years, we have expanded the questions that we address beyond chromatin – for example uncovering
novel mechanistic bases of aging and discovering new ways to extend lifespan. Similarly, inspired by our
recent finding that chromatin structure reduces the processing of DNA double-strand breaks to single-strand
DNA (termed DNA end resection), we have devised innovative CRISPR/Cas9 gRNA library screening
approaches to identify novel activities that regulate DNA end resection during DNA double-strand repair.
Most of the cells in the human body are in G0/G1-phase and it is critical that excessive DNA end resection
does not occur in these cells. If it were to occur, it would block DNA repair by the only pathway that is used to
repair DNA double-strand breaks in G0/G1-phase cells, namely non-homologous end joining (NHEJ), and it
would result in translocations and deletions from the ensuing homology-mediated repair. Indeed, the extent
of DNA end resection is the critical decision point in the choice between using the NHEJ or homologous
recombination (HR) pathway for repairing DNA double-strand breaks. We propose that mechanisms must be
in place that limit excessive DNA end resection in G0/G1-phase cells to prevent HR, yet enable sufficient DNA
end processing of un-ligatable DNA ends to allow NHEJ-mediated repair. The proteins and pathways that
regulate the extent of DNA end resection in G0/G1-phase cells are currently unknown. Thus, a major goal of
this program is to discover the machinery and mechanisms that regulate DNA end resection in G0/G1-phase
cells. We are uniquely positioned to do this, based on our expertise, novel genetic screening approach and
compelling preliminary data.
Another critical, yet poorly understood, aspect of genome maintenance is how gene expression is
“shut-off” in the vicinity of a DNA lesion to prevent collisions between the transcription and DNA repair
machinery. Similarly, it is crucial that transcription is restarting after DNA double-strand break repair, but the
mechanism is unknown. We have recently discovered some of the proteins involved using our novel assays
and genetic screens, so the second major goal of this program is to discover the fundamental mechanisms of
transcriptional shut-off and restart around DNA double-strand breaks.
期刊论文(0)
专著(0)
科研奖励(0)
会议论文
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批准号:10744971
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项目类别:
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资助金额:$53.5万
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财政年份:2023
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负责人:Jessica K Tyler
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依托单位:
2nd Biennial ASBMB - BSC Symposium on the Interplay between Epigenetic Regulation and Genome Integrity
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批准号:10540502
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资助金额:$0.96万
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财政年份:2022
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依托单位:
Novel pathways that regulate DNA double-strand break repair events in mammalian cells
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批准号:10557230
-
项目类别:
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资助金额:$42.38万
-
财政年份:2021
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负责人:Jessica K Tyler
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依托单位:
Novel pathways that regulate DNA double-strand break repair events in mammalian cells
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批准号:10093685
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项目类别:
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资助金额:$42.38万
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财政年份:2021
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FASEB's The Reversible Protein Acetylation in Health and Disease Conference
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批准号:10230422
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资助金额:$0.8万
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Discovering the molecular mechanisms that determine replicative lifespan
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批准号:9317795
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资助金额:$56.57万
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依托单位:
Chromatin Assembly Structure and Function
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批准号:7864485
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资助金额:$26.88万
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财政年份:2009
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负责人:Jessica K Tyler
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依托单位:
FASEB Summer Conference on Transcriptional Regulation During Cell Growth
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批准号:7484009
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项目类别:
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资助金额:$1.3万
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财政年份:2008
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负责人:Jessica K Tyler
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依托单位:
Chromatin's Role in Repair of Radiation-induced Damage
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批准号:6747500
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资助金额:$4.12万
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依托单位:
Chromatin's Role in Repair of Radiation-induced Damage.
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批准号:7210170
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资助金额:$26.86万
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Chromatin's Role in Repair of Radiation-induced Damage
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批准号:8266337
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资助金额:$26.76万
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Chromatin Assembly Structure and Function
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批准号:7321396
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资助金额:$30.34万
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Chromatin's Role in Repair of Radiation-induced Damage
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资助金额:$27.38万
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财政年份:2002
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负责人:Jessica K Tyler
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Chromatin's Role in Repair of Radiation-induced Damage
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资助金额:$4.37万
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Chromatin Assembly, Structure and Function
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Chromatin Assembly Structure and Function
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Chromatin Assembly Structure and Function
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Chromatin's Role in Repair of Radiation-induced Damage
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批准号:10529319
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Chromatin's Role in Repair of Radiation-induced Damage
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依托单位:
海外基金