Dynamics of DNA nicking and unwinding by the RepC–PcrA complex
Carolina Carrascoa✉, César L. Pastranaa, Clara Aicart-Ramosa, Sanford H. Leubab, Saleem Ahmad Khanc, Fernando Moreno‐Herreroa✉
- aDepartment of Macromolecular Structures, Centro Nacional de Biotecnología, CSIC, Darwin 3, 28049 Cantoblanco, Madrid, Spain
- bDepartments of Cell Biology and Bioengineering, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, 5117 Centre Avenue, Pittsburgh, PA 15213, USA
- cDepartment of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, PA 15219, USA
- ✉Corresponding author
Papers open inside SciRah from the original publisher or repository; SciRah never hosts them.
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.
TL;DRResults 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 bpS−1 is found in the absence of pausing. (Semantic Scholar)