Bypass Mechanisms in Eukaryotic Replication
真核复制中的旁路机制
基本信息
- 批准号:10500889
- 负责人:
- 金额:$ 36.06万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2022
- 资助国家:美国
- 起止时间:2022-09-01 至 2027-08-31
- 项目状态:未结题
- 来源:
- 关键词:BiochemicalBiochemistryBiophysicsBypassCell ExtractsCell divisionCell physiologyCellsCellularityChromatinChromosomal InstabilityChromosomesClosure by clampCommunitiesComplexConflict (Psychology)CoupledDNADNA DamageDNA biosynthesisDNA replication forkDepositionDiseaseEnsureEpigenetic ProcessFailureGenerationsGeneticGenetic TranscriptionGenomeGenomicsGoalsHistonesHoloenzymesHuman PathologyHybridsKnowledgeLabelLeadLesionMalignant NeoplasmsMediationMediator of activation proteinMedical ResearchMolecularMolecular ChaperonesMolecular MachinesMotionNucleosomesPathway interactionsPhosphotransferasesPolymeraseProcessProteinsRNARegulationReplication InitiationReplication OriginResearchS phaseSlideSystemTechnologyTranscriptional RegulationUbiquitinationdesigndisorder preventionds-DNAgenome integrityimprovedinsightintermolecular interactionpolypeptidepreservationpreventreconstitutionresponsesingle moleculesingle-molecule FRETspatiotemporalstructural biologytool
项目摘要
PROJECT SUMMARY
Chromosomes are copied by a complex holoenzyme called the replisome. Obstacles are routinely negotiated
by the replisome with auxiliary mechanisms, collectively called DNA damage tolerance pathways, that ensure
genomic integrity via on-the-fly remodeling. The aberrance of these pathways can lead to chromosome
instability and a broad range of diseases including cancer. The Schauer Lab’s long-term goal is to thus
understand the molecular basis for genetic and epigenetic fidelity, with the goal of improving the treatment
and/or prevention of diseases. In this proposal, the Schauer Lab will use a fully functional replisome
reconstituted from over 30 pure polypeptides to study how replisomes bypass obstacles that regularly occur in
the genome while enforcing genetic and epigenetic integrity across generations. They also propose to develop
whole cell lysate systems to establish active replication forks on double-stranded DNA at natural origins of
replication without the need for replication initiation on synthetic forks. DNA damage tolerance mechanisms will
be studied using biochemistry, single-molecule biophysics, and structural biology. The structural dynamics of
the S-phase damage response will be characterized, with a focus on the mediator kinase Mrc1 and the multiple
ways it regulates the elongating replisome. The Schauer Lab also proposes to study the spatiotemporal
mechanisms of rescue of lesion-stalled replisomes by translesion synthesis polymerases, and how both Mrc1
and ubiquitination of DNA sliding clamps regulates this response. When replicating chromatin, nucleosomes
present a strong block to replication fork progression in the absence of histone chaperones. The Schauer Lab
will study histone dynamics at the replication fork in reconstituted chromatin, with a focus on regulation of
histone deposition symmetry by histone chaperones and in the molecular mechanisms of various replication-
coupled histone chaperones themselves. Tools will be developed to track histone fate and dynamics at the
single-molecule level. The goal is to get a better understanding of the processes that control epigenetic
inheritance, important for maintaining cellularity during cell division. Finally, the Schauer Lab proposes to study
collisions between the replication machinery and an actively elongating transcription complex, since these
conflicts can be highly mutagenic. Transcriptional regulation by the rpb4/7 heterodimer will also be studied.
Transcription will be reconstituted from either purified proteins, or whole-cell extracts, or a combination of the
two. The Schauer Lab is developing biochemical and single-molecule tools for these projects that will afford an
unprecedented glimpse into the molecular mechanisms behind these critical processes. Single-molecule
fluorescence resonance energy transfer (smFRET) will be employed to track intermolecular interactions,
allowing a characterization of the structural dynamics of these systems. Further, technology will be developed
to track dynamic motions of fluorescently labeled proteins on double-tethered DNA at the single-molecule level.
The mechanistic insight afforded by these studies will be beneficial for the medical research community.
项目总结
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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Grant Schauer其他文献
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