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Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter
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A novel cell permeable DNA replication and repair marker.
Henry D Herce1, Malini Rajan, Gisela Lättig-Tünnemann
1a Department of Biology , Technische Universität Darmstadt ; Darmstadt , Germany.
Nucleus (Austin, Tex.)
|December 9, 2014
Summary
Researchers developed a cell-permeable peptide marker to track DNA replication and repair in live cells. This innovative tool bypasses transfection, offering a new way to study these essential cellular processes.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Proliferating Cell Nuclear Antigen (PCNA) is crucial for DNA replication and repair.
- Studying PCNA dynamics typically requires transfection with PCNA fusions, which is challenging in some cell types.
Purpose of the Study:
- To develop a cell-permeable marker for real-time visualization of DNA replication and repair.
- To create a tool that bypasses the need for cell transfection.
Main Methods:
- Engineered a peptide construct with three components: a PCNA-binding peptide, a cell-penetrating peptide (HIV-1 TAT), and an in vivo cleavable linker.
- Tested the construct's uptake and function in human, hamster, and mouse cells.
- Developed a peptide-based staining method as an alternative to PCNA antibodies.
Main Results:
- The peptide construct was rapidly internalized by various cell types.
- The construct effectively labeled PCNA at replication and repair sites in live cells.
- Demonstrated the first in vivo cell-permeable peptide marker for DNA replication and repair.
Conclusions:
- The developed peptide marker provides a versatile tool for instantaneous labeling of DNA repair and replication processes.
- This method offers an alternative to traditional PCNA antibody staining for immunofluorescence.
- The tool facilitates the study of fundamental cellular processes in both live and fixed cells.
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