Administrative Supplement to : Functional crosstalk between the Fanconi Anemia and ATRX/DAXX histone chaperone pathways
Administrative Supplement to : Functional crosstalk between the Fanconi Anemia and ATRX/DAXX histone chaperone pathways
批准号:
10387846
负责人:
Alexandra Theresia Sobeck
金额:
$5.92万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
2019
资助国家:
美国
项目状态:
已结题
起止时间:
2019-05-01 至 2023-04-30
关键词:
Administrative SupplementCRISPR/Cas technologyCancer cell lineCell LineCell SeparationCell SurvivalCellsDAXX geneDNA RepairDNA biosynthesisDNA replication forkFANCD2 proteinFanconi&aposs AnemiaGene TargetingGenesGenome StabilityHistonesHumanHuman Cell LineKnock-inKnock-outKnowledgeLaboratoriesLasersLinkMalignant NeoplasmsMediatingMicrofluidicsMolecularMolecular ChaperonesMolecular StructureNormal CellPathway interactionsRecoveryRoleS PhaseSorting - Cell MovementSyndromeSystemTestingalpha-Thalassemiabiological adaptation to stresscancer cellexperimental studyhomologous recombinationinsightloss of function mutationmutantnondeletion type alpha-thalassemia/mental retardation syndromeprotein complexreplication stressstem
中文摘要
项目总结
英文摘要
PROJECT SUMMARY
Studies from our laboratory provided evidence that the Fanconi Anemia (FA) and Alpha Thalassemia Retardation
Syndrome X-linked (ATRX) pathways are interconnected to promote genome stability in the face of DNA
replication stress by supporting homologous recombination (HR) mechanisms. Our previous observations led us
to hypothesized that ATRX cooperates with FANCD2 to promote HR-mediated DNA replication fork rescue, but
also possesses additional, independent activities to support a distinct subset of DNA repair steps.
So far, our study results were mostly obtained using human cancer-derived cell lines. Intriguingly, our recent
findings indicate that the FANCD2- and ATRX-dependent DNA replication stress responses activities differ
mechanistically between human cancer cells and non-cancer (“normal”) cells.
To test our hypothesis, we propose to investigate FANCD2 and ATRX functions in normal human cell lines,
namely RPE1 and BJ cells. To this end, we will generate normal human cells genetically null for ATRX,
FANCD2, or both, as well as cells carrying ATRX loss-of-function mutations. Importantly, efficient ATRX gene
knock-out or knock-in in normal human cells requires the use of a gentle cell sorter that (a) allows single live
cell sorting while maintaining high ATRX mutant cell viability and (b) allows for specific bulk purification of S-
phase cells to increase subsequent ATRX knock-in targeting efficiency. We propose to use a Sony benchtop
SH800 cell sorter equipped with two lasers and microfluidics sorting chips, in order to:
(1) Perform single live cell sorting of CRISPR/Cas9 targeted, ATRX- and ATRX/FANCD2-null RPE1 and
BJ cells
(2) Perform S-phase-specific bulk pre-sorting of RPE1 and BJ cells, followed by CRISPR/Cas9 mediated
ATRX gene knock-in.
Generating these mutant RPE1 and BJ cells utilizing the SONY SH800 sorter will then allow us to (i) elucidate
the molecular and structural makeup of ATRX-FANCD2 protein complexes; (ii) Determine molecular
mechanisms of ATRX/FANCD2-mediated replication fork recovery; (iii) Dissect FANCD2-dependent and -
independent roles of ATRX during DNA repair.
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会议论文
Functional crosstalk between the Fanconi Anemia and ATRX/DAXX histone chaperone pathways
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批准号:9919598
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项目类别:
-
资助金额:$32.23万
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财政年份:2019
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负责人:Alexandra Theresia Sobeck
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依托单位:
Role of EMSY protein complexes in the FA DNA repair pathway
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批准号:9100967
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项目类别:
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资助金额:$19.51万
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财政年份:2016
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负责人:Alexandra Theresia Sobeck
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依托单位:
海外基金