Bacillus subtilis RadA/Sms-Mediated Nascent Lagging-Strand Unwinding at Stalled or Reversed Forks Is a Two-Step Process: RadA/Sms Assists RecA Nucleation, and RecA Loads RadA/Sms
Abstract
:1. Introduction
2. Results
2.1. RadA/Sms Partially Counters the Negative Effect of SsbA on RecA Nucleation onto cssDNA
2.2. The RecO-SsbA Mediator Poorly Counteracts the Negative Effect of RadA/Sms on RecA ATPase
2.3. RadA/Sms Catalyzes Unwinding of Reversed Forks with Longer Nascent Lagging-Strand, but RecA and SsbA Reduce Unwinding
2.4. How RecA Activates RadA/Sms to Unwind a HJ Structure with a Nascent Leading-Strand Longer Than the Nascent Lagging-Strand
2.5. Two Step RadA/Sms Loading on the Nascent-Lagging Strand of HJ-Lead DNA
2.6. RecA Mediators Increase RecA Dynamics and Limit RadA/Sms Unwinding of Reversed Forks
2.7. RecA Activates RadA/Sms Unwinding of the Nascent Lagging-Strand of Synthetic Stalled Forks
3. Discussion
4. Materials and Methods
4.1. Strains and Plasmids
4.2. Enzymes, Reagents, Protein and DNA Purification, Protein–Protein Interaction
4.3. ATP Hydrolysis Assays
4.4. DNA Unwinding Assays
4.5. RecA-Mediated DNA Strand Exchange
4.6. Protein–Protein Interaction Assays
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Torres, R.; Carrasco, B.; Alonso, J.C. Bacillus subtilis RadA/Sms-Mediated Nascent Lagging-Strand Unwinding at Stalled or Reversed Forks Is a Two-Step Process: RadA/Sms Assists RecA Nucleation, and RecA Loads RadA/Sms. Int. J. Mol. Sci. 2023, 24, 4536. https://doi.org/10.3390/ijms24054536
Torres R, Carrasco B, Alonso JC. Bacillus subtilis RadA/Sms-Mediated Nascent Lagging-Strand Unwinding at Stalled or Reversed Forks Is a Two-Step Process: RadA/Sms Assists RecA Nucleation, and RecA Loads RadA/Sms. International Journal of Molecular Sciences. 2023; 24(5):4536. https://doi.org/10.3390/ijms24054536
Chicago/Turabian StyleTorres, Rubén, Begoña Carrasco, and Juan C. Alonso. 2023. "Bacillus subtilis RadA/Sms-Mediated Nascent Lagging-Strand Unwinding at Stalled or Reversed Forks Is a Two-Step Process: RadA/Sms Assists RecA Nucleation, and RecA Loads RadA/Sms" International Journal of Molecular Sciences 24, no. 5: 4536. https://doi.org/10.3390/ijms24054536