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. 2016 Oct 14;44(18):8885-8896.
doi: 10.1093/nar/gkw689. Epub 2016 Aug 3.

Force and twist dependence of RepC nicking activity on torsionally-constrained DNA molecules

Cesar L Pastrana  1 , Carolina Carrasco  1 , Parvez Akhtar  2 , Sanford H Leuba  3 , Saleem A Khan  2 , Fernando Moreno-Herrero  4
Affiliations

Force and twist dependence of RepC nicking activity on torsionally-constrained DNA molecules

Cesar L Pastrana et al. Nucleic Acids Res. .

Abstract

Many bacterial plasmids replicate by an asymmetric rolling-circle mechanism that requires sequence-specific recognition for initiation, nicking of one of the template DNA strands and unwinding of the duplex prior to subsequent leading strand DNA synthesis. Nicking is performed by a replication-initiation protein (Rep) that directly binds to the plasmid double-stranded origin and remains covalently bound to its substrate 5'-end via a phosphotyrosine linkage. It has been proposed that the inverted DNA sequences at the nick site form a cruciform structure that facilitates DNA cleavage. However, the role of Rep proteins in the formation of this cruciform and the implication for its nicking and religation functions is unclear. Here, we have used magnetic tweezers to directly measure the DNA nicking and religation activities of RepC, the replication initiator protein of plasmid pT181, in plasmid sized and torsionally-constrained linear DNA molecules. Nicking by RepC occurred only in negatively supercoiled DNA and was force- and twist-dependent. Comparison with a type IB topoisomerase in similar experiments highlighted a relatively inefficient religation activity of RepC. Based on the structural modeling of RepC and on our experimental evidence, we propose a model where RepC nicking activity is passive and dependent upon the supercoiling degree of the DNA substrate.

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Figures

Figure 1.
Figure 1.
General mechanism of plasmid rolling-circle replication (RCR). (Step I) A supercoiled plasmid containing the double-strand origin (dso) and single-strand origin (sso) is the substrate for DNA replication and is first nicked by a RepC dimer. A secondary structure at the dso facilitates binding and/or nicking of the DNA by RepC which becomes covalently attached to the 5′-P end of the DNA after nicking. (Step II) PcrA helicase interacts with RepC and unwinds the duplex DNA displacing the leading strand of the plasmid and leaving a free 3′-OH end at the dso. (Step III) DNA polymerase III loads at the free 3′end and synthesize a new leading strand. The displaced leading strand is protected by SSB proteins. (Step IV) A covalently closed single-stranded DNA is produced that can be subsequently converted to dsDNA which involves the synthesis of an RNA primer at the sso by the RNA polymerase followed by DNA synthesis by DNA polymerases I and III.
Figure 2.
Figure 2.
Nicking and religation activities of RepC probed by Magnetic Tweezers (MT). (A) DNA substrate employed in MT assays. The substrate contains two handles labeled with biotins and digoxigenins that bind to the magnetic bead and glass surface, respectively. The central part contains the nicking site at 1480 bps from the biotinylated DNA end. (B) MT assay. Torsionally-constrained DNA molecules are first negatively supercoiled by applying 30 negative (counter clockwise) rotations (step 1). RepC nicks the DNA and a full recovery of the original height is observed (step 2). To check if DNA molecules can be further coiled, i.e. they have been religated, positive rotations at low force are applied (step 3). (C) Extension, Turns and Force time courses that correspond to the supercoiling of torsionally-constrained DNA molecules. (D) Nicking activity of RepC. (E) RepC religated a fraction of the nicked molecules as they could be further supercoiled by application of positive rotations. Data were acquired at 60 Hz and filtered down to 3 Hz (displayed).
Figure 3.
Figure 3.
RepC nicking activity occurs only at negative rotations. (A) DNA was nicked only at negative turns of the magnet. First, the DNA molecule was positively supercoiled (+30 rotations) and the protein injected in the cell. Then, the molecule was untwisted and negatively supercoiled by applying from +30 to −30 turns. A nick event was detected (see arrow) at −7 turns. (B) An offset between magnet turns and linking number was taken into account to determine the ΔLk at which RepC nicked the DNA. Data were acquired at 60 Hz and filtered down to 3 Hz (both displayed). Magnets were rotated at 1 Hz. Force was 0.34 pN, constant in the experiment. (C) Histogram of ΔLk (N = 83). The Gaussian fit provides a mean value of ΔLk and supercoiling degree σ (quoted in the figure). (D) Normalized cumulative integral of the histogram representing the probability of nicking. Values of ΔLk and σ, quoted in the figure, relate to the characteristic 0.5 probability.
Figure 4.
Figure 4.
RepC nicking activity is force- and twist-dependent (A) Nicking probability as a function of linking number for a range of forces of 0.05–1.0 pN. Probabilities were determined as described in Figure 3. The nicking probability depends on the force applied to the DNA substrate, requiring a larger number of turns at low forces to observe RepC nicking. Also the distributions got sharper at higher forces. (B) Characteristic ΔLk (P = 0.5) for different forces. The line is a linear fit between 0.05 and 0.34 pN that provides a σ = −0.032 at zero force.
Figure 5.
Figure 5.
RepC–DNA interaction during nicking and religation activities. (A) Characteristic supercoils removal time course taken at 0.34 pN. Approximately 30 turns were released in 0.3 s. Data were acquired at 500 Hz. (B) Histogram of velocities of plectoneme release for RepC, hTopIB and Nb.BbvCI. Frequencies are fitted to a Gaussian function and the mean and s.e.m values are quoted in the figure. RepC showed an intermediate velocity between the topoisomerase and the nicking enzyme. (C) Nicking and religation experiment with RepC. RepC occasionally religated the nicked DNA. This is shown as a dip in the extension while the magnets continuously rotate in a negative direction. (D) RepC cannot nick positively supercoiled DNA. (E and F) Nicking and religation experiment with hTopIB. hTopIB nicks and religates efficiently while the magnets continuously rotate in either negative or positive direction.
Figure 6.
Figure 6.
Passive nicking model for RepC. (A) Phyre2 prediction of the structure of RepC based on its 80% sequence homology with RepD (PDB entry 4CWC). RepC is a dimer with separate DNA binding (blue) and nicking domains (arrows). It has a C-shape morphology with a cavity of 30-40 Å, enough to accommodate the DNA for cleavage. (B) RepC binds to linear DNA but does not nick it because the cruciform structure is not induced. Application of a twist in a torsionally-constrained linear DNA produces extrusion of the cruciform and places the tip of the hairpin at the catalytic site of RepC resulting in efficient nicking. Nicking of linear DNA is inefficient as it would require sliding of the molecule through the cavity of RepC or direct binding to the nicking site. (C) A negatively supercoiled plasmid already contains the appropriate cruciform structure and is a substrate for RepC binding and efficient nicking.
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