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Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry
Published on: August 19, 2012
Generation of a mono-ubiquitinated PCNA mimic by click chemistry
Silvia Eger1, Benoît Castrec, Ulrich Hübscher
1Department of Chemistry, University of Konstanz, Universitätsstrasse 10, 78457 Konstanz, Germany.
Abstract:
Genotoxic stress results in more than 50 000 damaged DNA sites per cell per day. During DNA replication, processive high-fidelity DNA polymerases generally stall at DNA lesions and have to be displaced by translesion synthesis DNA polymerases, which are able to bypass the lesion. This switch is mediated by mono-ubiquitination of the processivity factor proliferating cell nuclear antigen (PCNA). To further investigate the regulation of the DNA polymerase exchange, we developed an easy and efficient method to synthesize site-specifically mono-ubiquitinated PCNA by click chemistry. By incorporating artificial amino acids that carry an azide (Aha) or an alkyne (Plk) in their side chains, into ubiquitin (Ub) and PCNA, respectively, we were able to link the two proteins site-specifically by the Cu(I) -catalyzed azide-alkyne cycloaddition. Finally, we show that the synthetic PCNA-Ub is able to stimulate DNA synthesis by DNA polymerase δ, and that DNA polymerase η has a higher affinity for PCNA-Ub than to PCNA.
Insights
Researchers created a new method to synthesize mono-ubiquitinated proliferating cell nuclear antigen (PCNA), a key protein in DNA repair. This synthetic PCNA-Ub aids DNA synthesis and shows altered polymerase interactions, advancing DNA damage response studies.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Genotoxic stress causes extensive DNA damage daily, necessitating efficient DNA repair mechanisms.
- DNA replication involves a switch from high-fidelity polymerases to translesion synthesis polymerases at DNA lesions.
- This switch is regulated by the mono-ubiquitination of proliferating cell nuclear antigen (PCNA).
Purpose of the Study:
- To develop an efficient method for synthesizing site-specifically mono-ubiquitinated PCNA.
- To investigate the regulation of DNA polymerase exchange during DNA repair.
Main Methods:
- Utilized click chemistry, specifically copper(I)-catalyzed azide-alkyne cycloaddition.
- Incorporated artificial amino acids (azide-bearing in ubiquitin and alkyne-bearing in PCNA) for site-specific protein conjugation.
- Synthesized mono-ubiquitinated PCNA (PCNA-Ub).
Main Results:
- Demonstrated the successful synthesis of site-specifically mono-ubiquitinated PCNA using click chemistry.
- Showed that synthetic PCNA-Ub stimulates DNA synthesis by DNA polymerase δ.
- Observed a higher affinity of DNA polymerase η for PCNA-Ub compared to unmodified PCNA.
Conclusions:
- The developed click chemistry approach provides an efficient way to create modified PCNA for studying DNA repair.
- Mono-ubiquitinated PCNA plays a crucial role in modulating DNA polymerase activity during replication stress.
- Findings offer insights into the mechanisms of DNA damage tolerance and repair pathway regulation.
