Dynamics of DNA Nicking and Unwinding by the RepC-PcrA Complex

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.Ministerio de Economía y Competitividad (MINECO) [BFU2017-83794-P (AEI/FEDER, UE) to F.M.-H.]; European Research Council (ERC) under the European Union Horizon 2020 research and innovation grant agreemen t[681299 to F.M.-H.]; National Science Foundation [toS.H.L.]. C.C. was supported by a ‘Severo Ochoa’ postdoc-toral contract from the National Center of Biotechnology (CNB-CSIC); Funding for open access charge: EuropeanResearch Council [681299].Peer reviewe

Understanding the paradoxical mechanical response of in-phase A-tracts at different force regimes

A-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions i n v i vo . The crystallographic structure of A-tracts has been well characterized. However, the mechanical properties of these sequences is controversial and their response to force remains unexplored. Here, we rationalize the mechanical properties of in-phase A-tracts present in the C a e n o r h a b d i t i s e l e g a n s genome over a wide range of external forces, using single-molecule experiments and theoretical polymer models. Atomic Force Microscopy imaging shows that A-tracts induce long-range (∼200 nm) bending, which originates from an intrinsically bent structure rather than from larger bending flexibility. These data are well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments show that the mechanical response of A-tracts and arbitrary DNA sequences have a similar dependence with monovalent salt supporting that the observed A-tract bend is intrinsic to the sequence. Optical tweezers experiments reveal a high stretch modulus of the A-tract sequences in the enthalpic regime. Our work rationalizes the complex multiscale flexibility of A-tracts, providing a physical basis for the versatile character of these sequences inside the cell.The authors acknowledge the computer resources, technical expertise and assistance provided by the Red Española de Supercomputacion at the Minotauro Supercomputer (BSC, Barcelona). We thank Andrew Fire (Stanford University, USA) and Ralf Seidel (University of Leipzig, Germany) for providing us biological material required for the fabrication of the DNA molecules.Peer reviewe