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Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions
Published on: November 12, 2017
Protein Dynamics in Complex DNA Lesions
Radoslav Aleksandrov1, Anton Dotchev1, Ina Poser2
1Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str. Bl.21, 1113 Sofia, Bulgaria.
Abstract:
A single mutagen can generate multiple different types of DNA lesions. How different repair pathways cooperate in complex DNA lesions, however, remains largely unclear. Here we measured, clustered, and modeled the kinetics of recruitment and dissociation of 70 DNA repair proteins to laser-induced DNA damage sites in HeLa cells. The precise timescale of protein recruitment reveals that error-prone translesion polymerases are considerably delayed compared to error-free polymerases. We show that this is ensured by the delayed recruitment of RAD18 to double-strand break sites. The time benefit of error-free polymerases disappears when PARP inhibition significantly delays PCNA recruitment. Moreover, removal of PCNA from complex DNA damage sites correlates with RPA loading during 5'-DNA end resection. Our systematic study of the dynamics of DNA repair proteins in complex DNA lesions reveals the multifaceted coordination between the repair pathways and provides a kinetics-based resource to study genomic instability and anticancer drug impact.
Insights
DNA repair pathways coordinate complex lesion repair through precise protein timing. Error-prone polymerases are delayed, ensuring genomic stability and impacting cancer drug development.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Mutagens create diverse DNA lesions, necessitating complex repair mechanisms.
- Cooperation among DNA repair pathways for complex lesions is not well understood.
Purpose of the Study:
- To investigate the kinetics of DNA repair protein dynamics at complex DNA damage sites.
- To elucidate the coordination between different DNA repair pathways.
Main Methods:
- Measured and modeled recruitment/dissociation kinetics of 70 DNA repair proteins.
- Utilized laser-induced DNA damage in HeLa cells.
- Analyzed protein dynamics using clustering and kinetic modeling.
Main Results:
- Revealed distinct recruitment timescales for error-prone vs. error-free polymerases.
- Demonstrated delayed RAD18 recruitment to double-strand breaks impacts polymerase choice.
- Showed PCNA (Proliferating Cell Nuclear Antigen) removal correlates with RPA (Replication Protein A) loading during DNA resection.
Conclusions:
- The study provides a kinetic framework for understanding DNA repair pathway coordination.
- Findings offer insights into genomic instability mechanisms.
- Results can inform the development of anticancer drugs targeting DNA repair.
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