FtsK-dependent XerCD-dif recombination unlinks replication catenanes in a stepwise manner - PubMed (original) (raw)

Koya Shimokawa et al. Proc Natl Acad Sci U S A. 2013.

Abstract

In Escherichia coli, complete unlinking of newly replicated sister chromosomes is required to ensure their proper segregation at cell division. Whereas replication links are removed primarily by topoisomerase IV, XerC/XerD-dif site-specific recombination can mediate sister chromosome unlinking in Topoisomerase IV-deficient cells. This reaction is activated at the division septum by the DNA translocase FtsK, which coordinates the last stages of chromosome segregation with cell division. It has been proposed that, after being activated by FtsK, XerC/XerD-dif recombination removes DNA links in a stepwise manner. Here, we provide a mathematically rigorous characterization of this topological mechanism of DNA unlinking. We show that stepwise unlinking is the only possible pathway that strictly reduces the complexity of the substrates at each step. Finally, we propose a topological mechanism for this unlinking reaction.

Keywords: DNA topology; Xer recombination; band surgery; tangle method; topology simplification.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Proposed stepwise unlinking by XerCD-_dif_–FtsK recombination: Parallel RH 2m-cats [e.g., T(2,6)p] are converted to RH torus knots [e.g., T(2,5)] with directly repeated sites; such knots are converted to RH cats, and so on, iteratively, until the reaction stops at two open circles. (In ref. , RH torus links with parallel sites and up to 14 crossings, also called parallel 2m-cats and denoted by T(2,2m)p, were used as substrates of Xer recombination.)

Fig. 2.

Fig. 2.

(A) XerCD–FtsK-dif complex before recombination is modeled by the sum of tangles Ob + P. There, P encloses the core regions of the recombination sites and therefore can be seen as a ball with two very short DNA strings. Site-specific recombination occurs inside P. The action of cleavage and strand exchange is modeled by converting P into a tangle R. In the case of unknotted substrates, the two DNA strings outside the enzymatic complex have been observed to be supercoiled but not intertwined. However, in the case of knotted or linked substrates one would expect the DNA outside XerCD–FtsK not to be topologically trivial. For simplicity, we push any such topological complexity into the shaded area of the tangle in the figure, which we then refer to as the outside tangle O. We define O = Of + Ob, where Of includes the DNA outside XerCD-_dif_-FtsK. The recombination event is represented as a system of equations N(O + P) = substrate and N(O + R) = product, and the tangle O is not changed during the recombination. (B) Orientations of the recombination sites induce orientations to the strings inside P and R. When the two sites are inside P = (0) in parallel alignment, then R = (−1) or (1). When the sites are in P = (0) in antiparallel alignment, then R = (0,0). Tangles (0), (0,0), (−1), and (1) are the trivial tangles.

Fig. 3.

Fig. 3.

Unlinking pathway of RH 2m-cat: RH 2m-cat, RH (2m − 1)-torus knot, RH (2m − 2)-cat,…, RH trefoil, 2-cat, the unknot, trivial link.

Fig. 4.

Fig. 4.

Last three steps of DNA unlinking by XerCD–FtsK. At the top are the last three steps in the DNA unlinking pathway. Each of the three rows below shows one of the three possible topological mechanisms by which a trefoil is converted to a 2-cat, a 2-cat to an unknot, and an unknot to an unlink. Locally, the first mechanism, where P = (0) parallel and R = (−1), is consistent with the mechanism proposed for XerCD at psi (17). The other two pathways (rows 2 and 3) can be obtained by rigid rotation of the pathway in row 1, and correspond to P = (0) antiparallel and R = (0,0), and to P = (0) parallel and R = (1), respectively. The rotation axes are m1 and m2, as in Fig. 5. These mechanisms are consistent with the three proposed for Xer at psi (15).

Fig. 5.

Fig. 5.

Local site alignment. Due to a high level of sequence homology, we here assume that the XerCD–DNA synapse can be modeled using the data for Cre–DNA. This diagram is based on the crystal structure of Cre–DNA from ref. , in which the recombinase core complex is slightly off-planar, so that the cores appear either parallel or antiparallel in different projections. To improve clarity, the angles are doubled in this diagram. Let m2 be the horizontal axis (dashed line), and let m1 be the vertical axis (solid line). When looking down m2, the two sites appear in antiparallel alignment, whereas when seen from the reader’s perspective, the sites are in parallel. A rigid left-hand rotation of the DNA conformation around the axis m1 will result in string IV crossing over string I (15). Then the sites appear in antiparallel alignment and one crossing is added to the domain. Further left-hand rotation around the m2 axis results in III crossing over II and the sites returning to a parallel alignment.

Fig. 6.

Fig. 6.

(A) Two rational tangles and their Conway vectors. (B) Tangle addition of rational tangles A = (−5,0) and B = (−1). A + B is equivalent to the tangles ((\x{2212}5,\x{2212}1) and (6,\x{2212}1,0)). (C) N(A + B) is a 6-crossing RH torus link (RH 6-cat).

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