Characterization of LINE-1 reverse transcriptase activity
LINE-1 逆转录酶活性的表征
基本信息
- 批准号:10604881
- 负责人:
- 金额:$ 2.29万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2023
- 资助国家:美国
- 起止时间:2023-02-02 至 2023-05-11
- 项目状态:已结题
- 来源:
- 关键词:AgingAmino AcidsAutoimmune DiseasesBiochemicalBiological AssayBombyx moriCancer BiologyCellsChromosomal RearrangementClinicalCodeColon CarcinomaCytoplasmDNADNA DamageDNA NucleotidylexotransferaseDNA RepairDNA Sequence RearrangementDataDegenerative DisorderDiseaseDissociationEvolutionGelGenetic TranscriptionGenomeGenomicsGoalsHumanHuman GenomeHydrogen BondingImmune signalingIn VitroInterferonsIntronsL1 ElementsLabelLengthLesionLifeLife Cycle StagesLong Interspersed ElementsMacular degenerationMalignant NeoplasmsMalignant neoplasm of esophagusMeasuresMediatingModificationNucleic AcidsNucleosidesNucleotidesOpen Reading FramesPattern recognition receptorPhenotypePhylogenetic AnalysisPolymerasePolymersProcessProliferatingPropertyProteinsPseudouridineRNARNA BindingRNA CapsRNA Polymerase IIRNA ProcessingRNA vaccineRNA-Binding ProteinsRNA-Directed DNA PolymeraseReactionReadingResearchResearch ProposalsResolutionRetroelementsRetrotranspositionRetrotransposonReverse Transcriptase InhibitorsReverse TranscriptionRibonucleotidesRoleSeriesShort Interspersed Nucleotide ElementsSignal TransductionStructureSystemic Lupus ErythematosusTertiary Protein StructureTestingThumb structureTranscriptWorkclinically significantdriving forceexperimental studyhuman diseaseimprovedin vitro activityinsightinterestknowledge basemalignant breast neoplasmpolymerizationpseudotoxoplasmosis syndromepublic health researchrepaired
项目摘要
Project Summary
The objective of this research proposal is to characterize the enzymatic activities of the reverse transcriptase
(RT) of the human long interspersed element 1 (LINE-1, L1 RT). LINE-1 sequences constitute 17% of the
human genome, and L1 RT activity is responsible for approximately 40% of our genome because it has also
led to the proliferation of short interspersed elements (e.g., Alu), and other retroelements. L1 RT activity is also
implicated as a driving force behind a variety of human diseases such as macular degeneration, Aicardi-
Goutières syndrome, and systemic lupus erythematosus. LINE-1 expression and retrotransposition are
commonplace in numerous cancers, including 90-100% of breast, colon, and esophageal cancers, making
understanding the basic biochemical activity of L1 RT relevant to public health research.
Reverse transcription is key to the ability of LINE-1 to self-propagate in our genome, through a process
known as target-primed reverse transcription (TPRT). L1 RT is encoded by the second open reading frame
(ORF2) of L1, residing in the protein known as ORF2p. In this proposal, I show that I have isolated the ORF2p
RT domain, and that this has RT activity in vitro. I intend to use this protein domain, as well as full-length
ORF2p, and L1 RNPs to fully characterize the biochemical and enzymatic properties of L1 RT. In my first
Specific Aim, I will characterize its processivity, which is an indication of whether the RT stops and pauses, or
whether it keeps going until it reaches the end of a template. I will also measure the fidelity of L1 RT, which
defines how faithfully it copies DNA from an RNA template. Finally, I present preliminary sequencing data
showing that L1 RT has the unexpected ability to begin processing an RNA template without a primer and
propose rigorous experiments to study this feature. In my second Specific Aim, I focus on what happens when
L1 RT reaches the end of its template, which may be crucial to understanding how cells repair intermediates of
transposition. I present preliminary data showing that L1 RT adds extra nucleotides to the end of its cDNA and
propose studies to fully characterize this activity. Additionally, I will study the ability of L1 RT to switch to a
different template at the end of one template and whether LINE-1 RT can use modified RNA as a template,
such as the pseudouridine found in mRNA vaccines or the naturally occurring N6-methyladenosine
modification.
This work will advance our understanding of how LINE-1 operates inside cells and how LINE-1
completes its life cycle. The proposed research will have implications for cancer biology and innate immune
signaling where L1 RT is increasingly recognized as a DNA damaging agent and trigger for pattern recognition
receptors, respectively. Finally, this research will give insight into the evolution of LINE-1-like retroelements
that are found across all kingdoms of life.
项目总结
项目成果
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