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Updated: Dec 31, 2025
Author Spotlight: Unraveling the Dynamics of Eukaryotic DNA Replication Through Single-Molecule Visualization
Published on: September 27, 2024
Dynamics of DNA nicking and unwinding by the RepC-PcrA complex
Carolina Carrasco1, Cesar L Pastrana1, Clara Aicart-Ramos1
1Department of Macromolecular Structures, Centro Nacional de Biotecnología, CSIC, Darwin 3, 28049 Cantoblanco, Madrid, Spain.
Abstract:
The rolling-circle replication is the most common mechanism for the replication of small plasmids carrying antibiotic resistance genes in Gram-positive bacteria. It is initiated by the binding and nicking of double-stranded origin of replication by a replication initiator protein (Rep). Duplex unwinding is then performed by the PcrA helicase, whose processivity is critically promoted by its interaction with Rep. How Rep and PcrA proteins interact to nick and unwind the duplex is not fully understood. Here, we have used magnetic tweezers to monitor PcrA helicase unwinding and its relationship with the nicking activity of Staphylococcus aureus plasmid pT181 initiator RepC. Our results indicate that PcrA is a highly processive helicase prone to stochastic pausing, resulting in average translocation rates of 30 bp s-1, while a typical velocity of 50 bp s-1 is found in the absence of pausing. Single-strand DNA binding protein did not affect PcrA translocation velocity but slightly increased its processivity. Analysis of the degree of DNA supercoiling required for RepC nicking, and the time between RepC nicking and DNA unwinding, suggests that RepC and PcrA form a protein complex on the DNA binding site before nicking. A comprehensive model that rationalizes these findings is presented.
Insights
Researchers used magnetic tweezers to study how proteins RepC and PcrA interact during plasmid replication in bacteria. They found these proteins likely form a complex before nicking DNA, a key step in replication.
Area of Science:
- Molecular Biology
- Biochemistry
- Microbiology
Background:
- Rolling-circle replication is vital for plasmids with antibiotic resistance genes in Gram-positive bacteria.
- Replication initiation involves a replication initiator protein (Rep) and PcrA helicase for DNA unwinding.
- The precise interaction mechanism between Rep and PcrA during nicking and unwinding remains unclear.
Purpose of the Study:
- To investigate the interaction between Staphylococcus aureus plasmid pT181 initiator RepC and PcrA helicase.
- To elucidate the relationship between RepC's nicking activity and PcrA helicase unwinding using magnetic tweezers.
- To develop a comprehensive model for the RepC-PcrA protein complex in DNA replication.
Main Methods:
- Utilized magnetic tweezers to monitor PcrA helicase unwinding dynamics.
- Analyzed PcrA translocation rates and processivity with and without pausing.
- Investigated the effect of single-strand DNA binding protein on PcrA activity.
- Assessed DNA supercoiling requirements for RepC nicking and temporal coupling with unwinding.
Main Results:
- PcrA exhibits high processivity as a helicase, with average translocation rates of 30 bp/s, reduced by pausing.
- PcrA translocation velocity was unaffected by single-strand DNA binding protein, but processivity slightly increased.
- RepC nicking requires specific DNA supercoiling, and its timing suggests a pre-nicking RepC-PcrA complex formation.
Conclusions:
- RepC and PcrA likely form a stable complex at the DNA binding site prior to nicking.
- This interaction is crucial for the coordinated initiation of rolling-circle replication.
- A detailed model explaining the interplay between RepC nicking and PcrA unwinding is proposed.
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