Closing the DNA replication cycle: from simple circular molecules to supercoiled and knotted DNA catenanes

Abstract

Due to helical structure of DNA, massive amounts of positive supercoils are constantly introduced ahead of each replication fork. Positive supercoiling inhibits progression of replication forks but various mechanisms evolved that permit very efficient relaxation of that positive supercoiling. Some of these mechanisms lead to interesting topological situations where DNA supercoiling, catenation and knotting coexist and influence each other in DNA molecules being replicated. Here, we first review fundamental aspects of DNA supercoiling, catenation and knotting when these qualitatively different topological states do not coexist in the same circular DNA but also when they are present at the same time in replicating DNA molecules. We also review differences between eukaryotic and prokaryotic cellular strategies that permit relaxation of positive supercoiling arising ahead of the replication forks. We end our review by discussing very recent studies giving a long-sought answer to the question of how slow DNA topoisomerases capable of relaxing just a few positive supercoils per second can counteract the introduction of hundreds of positive supercoils per second ahead of advancing replication forks.

Figure 1.

Crossing sign convention, linking number (Lk) and writhe (Wr). (A) Schematic presentation of a double-stranded DNA minicircle. For topological considerations both strands of DNA are given the same direction along the circle. Therefore, for right-handed DNA helix the intra-duplex inter-strand crossings have positive signs. The linking number (Lk) of the presented DNA minicircle is 7 (see the main text). The twist (Tw) of that minicircle is also 7 as the helix makes 7 complete right-handed turns. (B) Crossings between two oriented segments can be only of positive or negative sign. Crossing has negative sign if the direction arrow that is closer to the observer would need to be turned in a clockwise direction to overly it with the direction arrow that is further from the observer. If that direction of turning is counter-clockwise, the crossing has positive sign. Of course, the turning angle cannot be larger than 180°. (C) In negatively supercoiled DNA molecule forming a regular superhelix, the self-crossings of DNA axis have negative signs. Notice that orientation of the underlying and overlying direction arrows at each crossing are not independent from each other but result from assigning a consistent direction (see the black arrows) along the whole DNA molecule analyzed. The DNA double-helix is shown in blue and green.

Figure 2.

Concepts of ΔLk, ΔTw and ΔWr and interconversions between Wr and Tw in covalently closed DNA molecules. (A and B) When relaxed circular DNA is unwound by 5 turns at the site of the nick and then ligated the molecule becomes negatively supercoiled with ΔLk = −5. The torsional stress caused by unwinding is redistributed into changes of writhe (ΔWr) and twist (ΔTw). Where the Δ indicates the difference of Wr or Tw values, respectively, between torsionally relaxed form and the supercoiled form of otherwise identical DNA molecules. Due to a specific ratio between bending and torsional elasticity of DNA, ca 70–80% of ΔLk is converted into ΔWr. The rest of (ΔLk = −5) goes into decrease of DNA helicity (ΔTw ≈ −1). (B and C) In negatively supercoiled DNA, the strand separation during initiation of DNA replication is facilitated as it relaxes negative supercoils and thus decreases the elastic energy of the DNA molecules. In the shown case, upon strand separation of 5 turns of DNA helix the molecule reached the state with ΔWr = 0 and ΔTw = ΔLk i.e. the state that minimizes the elastic energy of circular DNA molecules. (C and D) When DNA replication continues in the absence of DNA topoisomerases, the DNA molecules become positively supercoiled and their elastic energy grows, which at some point will block a further progression of DNA replication. In the shown case, the strand separation extending over 6 additional turns of DNA helix resulted in the decrease of Tw by only 5 units as freshly replicated duplex portions form right-handed precatenanes permitting parental strands to twist once around the imaginary axis of the molecule. Therefore, the molecule reached the state with ΔWr = 4, ΔTw = −9 and ΔLk = −5. Notice that in the absence of DNA topoisomerases, the ΔLk stays constant.

Figure 3.

DNA topoisomerases. (A) Type IA DNA topoisomerases (topo IA) relax negatively supercoiled DNA regions that are excessively supercoiled and thus have their DNA helix destabilized. Binding of topo IA topoisomerases provokes further DNA destabilization and local strand separation. Separated strands, which are torsionally very flexible, start winding around each other in a left-handed way (L-DNA) as this partially releases the torsional stress in negatively supercoiled DNA. Topo IA introduces a transient cut into one strand in the destabilized region that winds in a left-handed way. The energy of opened phospho-diester bond is conserved by formation of a covalent bond involving 5′-end of the cut strand and one of tyrosines of topo IA. Subsequently, topo IA passes the intact strand of destabilized duplex through the opening in the cut strand. The continuity of strands is re-established by replacing the covalent bond connecting 5′-end of the cut strand with topo IA by a phospho-diester bond connecting it with 3′-end of the cut strand. The entire process results in the increase of the linking number by one, which decreases the excessive torsional stress in a negatively supercoiled DNA. (B) Type IB topoisomerases (topo IB) create transient swivels in DNA by cutting one strand and letting the DNA to swivel around the uncut strand (single chemical bonds can undergo unrestricted axial rotation). The region where DNA swiveling occurs is enclosed within the enzyme and since this enclosure limits the speed of torsional relaxation, the process is called controlled rotation. The energy of the cut phospho-diester bond is conserved by formation of a covalent bond involving 3′ end of the cut strand and one of tyrosines of topo IB. After a brief period of controlled rotation, the continuity of the cut strand is re-established by replacing the covalent bond connecting 3′-end of the cut strand with topo IB by a phospho-diester bond connecting it with 5′-end of the cut strand. The entire process results in the increase or decrease of the linking number by one or more units and can decrease torsional stress in negatively or positively supercoiled DNA regions. Panels (A) and (B) are based on Figure 1 in (32), where more details about mechanisms of DNA topoisomerases can be found. (C) Type II DNA topoisomerases transiently cut one duplex DNA region and move another duplex DNA region through the transient opening. Each passage changes the writhe by two units and this results in changing the linking number by two units. In principle, each passage can decrease or increase the linking number by two units. However, most of type II DNA topoisomerases preferably act on DNA crossings that have the geometry characteristic for intramolecular crossings with positive sign. Action of type II DNA topoisomerase at such crossings decreases the linking number of affected DNA molecules. The decrease of Lk is important for the relaxation of positive supercoiling generated during DNA replication and is also essential for the introduction of negative supercoiling in bacteria by DNA gyrase that is one of type II topoisomerases in bacteria.

Figure 4.

Changes of DNA topology during replication. (A) In most prokaryotes, native unreplicated molecules are negatively supercoiled. (B) Negative supercoiling facilitates the opening of the double-helix required for transcription and replication to begin and advance. This opening, however, generates positive torsional tension ahead of the fork. At the beginning, when unreplicated portions are sufficiently large, several molecules of DNA gyrase acting independently from each other can eliminate all positive torsional tension and even maintain negative supercoiling in the yet unreplicated portion of the molecules. Circular red arrow indictes that freshly replicated portions have the freedom of axial rotation due to the presence of single-stranded regions at replication forks.(C) As replication advances and there is less place for topoisomerases to act ahead of the replication fork, positive supercoiling accumulates in the unreplicated region. Rotations of the forks, indicated with a blue circular arrow, partially releases the torsional stress by formation of precatenanes that wind around each other in a right-handed way and have positive signs of their crossings as the direction of newly replicated portions of the molecules follows the direction of parental strands. (D) Once the replication is completed, fully replicated molecules form right-handed catenanes in which individual DNA circles are multiply catenated with each other. The sign of catenane crossings is positive as newly replicated molecules inherit the directions of parental strands. The unreplicated parental DNA double-helix is shown in blue and green while newly synthesized strands are depicted in red.

Figure 5.

Simulation snapshot of negatively supercoiled postreplicative catenanes. The only left-handed crossing (indicated with an arrow) naturally forms at the place where topoisomerases preferentially acting at left-handed crossings can efficiently decatenate postreplicative catenanes. The insets schematically show crossings with left- and right-handed geometry. Left-handed crossings are these where one would need to turn the overlying segment counter-clockwise to make this segment perpendicular to the underlying segment. In right-handed crossings, the required rotation is in a clockwise direction. Of course, the required rotations can’t exceed 90°.

Figure 6.

Replication fork regression and its reversal. (A) When the activities of cellular topoisomerases are perturbed, the positive supercoiling generated by ongoing DNA replication is not relaxed quickly enough. The resulting mechanical stress causes then the formation of positive supercoiling in the yet unreplicated portion of the DNA and of positive windings of the freshly replicated regions around each other. (B) Once the mechanical stress reaches a critical value, the replication is stopped and replisomes that normally prevent the parental strands from re-annealing are likely to be dislodged. At this point, mechanical stress resulting from positive supercoiling can be relaxed by the regression of one of the replication forks. (C) Action of DNA gyrase on DNA molecules with reversed forks causes the regression of fork reversal. In negatively supercoiled DNA molecules, it is energetically favorable to increase the region over which the parental DNA strands are separated and this can be achieved by the reversal of fork regression.

Figure 7.

Different forms of DNA catenanes. (A) CatA catenanes composed of two fully replicated, torsionally relaxed rings that are singly interlinked and show two positive intermolecular crossings. (B) CatA catenanes composed of two torsionally relaxed rings that are interlinked twice and show four positive intermolecular crossings. (C) CatA catenanes composed of two fully replicated and torsionally relaxed rings that are interlinked three times with each other and show six positive intermolecular crossings. Notice that both rings wind around a common axis. (D) CatB catenane composed of one negatively supercoiled DNA molecule with four intramolecular crossings that is interlinked twice with torsionally relaxed DNA ring. Notice that the effective ΔLk (ΔLk_e) of the left ring is approximately −4 while for the right ring, ΔLk_e = 0. (E) CatC catenane composed of two negatively supercoiled DNA rings that are interlinked twice with each other. Each ring shows four negative intramolecular crossings and contribute to four positive inter-molecular crossings. Notice that in this case, for both rings, ΔLk_e amounts to approximately −4. (a) and (b) denote the individual rings. The parental chains are in blue and green while newly synthesized chains are depicted in red.

Figure 8.

DNA knots and knotted bubbles. Cartoons representing nicked DNA rings forming various knots. (A) An unknotted molecule. (B) The classical trefoil knot with three positive nodes. (C) The so-called ‘figure of 8’ knot with two positive and two negative nodes that make this knot achiral. (D) A knotted molecule with five positive crossings. (E) A knotted molecule showing four positive and two negative nodes. (F) Another knotted molecule with six crossings but showing four negative and two positive nodes. Signs of the crossings are determined using the convention shown in Fig. 1B. (G) A partially replicated DNA molecule with a nick at the unreplicated region containing an inter-chromatid trefoil knot in the replicated region. (H) A partially replicated DNA molecule with a nick at the unreplicated region containing an intra-chromatid trefoil knot in the replicated region. The parental chains are in blue and green while newly synthesized chains are depicted in red. We used here Alexander-Briggs notation of knots (2). The notation is composed of two numbers. The first number indicates a minimal number of crossing a given knot can have and the second number written as subscript indicates the tabular position of a given knot among knots with a given number of crossings. Thus, for example, the notation 6₂ indicates that the minimal number of crossings of this knot is 6 and its image can be found in topological tables of knots at the second position among knots with six crossings.

Figure 9.

Supercoiled and knotted catenanes. (A) One fully replicated covalently closed and negatively supercoiled ring interlinked once with another fully replicated and relaxed ring. (B) Two fully replicated, covalently closed and positively supercoiled sister duplexes interlinked once. (C) A fully replicated and relaxed ring harboring a trefoil knot interlinked once with another fully replicated and relaxed ring. (D) Two fully replicated and relaxed sister duplexes both harboring a trefoil knot, interlinked once. (E) One fully replicated and relaxed ring harboring a trefoil knot interlinked once with another fully replicated covalently closed and negative supercoiled ring. (F) One fully replicated covalently closed and negative supercoiled ring harboring a trefoil knot interlinked once with another fully replicated covalently closed and negative supercoiled ring devoid of knots. (a) and (b) denote the individual rings. The parental chains are in blue and green while newly synthesized chains are depicted in red.

Figure 10.

Topological transitions involved in closing the replication cycle of negatively supercoiled circular DNA molecules. The cycle marked with green arrows constitute a predominant ‘topology cursus’ taken by the majority of replicating circular DNA molecules such as bacterial plasmids. Gray arrows indicate facultative ‘topology cursi’ taken by part of replicating DNA molecules. (A) Not replicating, negatively supercoiled DNA molecule. (B) DNA molecule that initiated its replication. Negative supercoiling helps to initiate strand separation and continuous action of several DNA gyrase molecules acting within relatively large unreplicated portion of DNA molecule assures that the molecule can maintain its negative supercoiling, which favors further strand separation and relaxes positive supercoiling generated by strand separation. (C) DNA molecule toward the end of replicative strand separation. Unreplicated portion is too small to allow DNA gyrase(s) to bind and to relax positive supercoiling generated by replicative strand separation. Accumulating positive supercoiling induces formation of precatenanes that wind around each other in a right-handed sense and have positive signs of their crossings. (D) Freshly replicated DNA in which right-handed precatenane windings are converted to right-handed catenanes windings. (E) Once the continuity of freshly synthesized strands is achieved, each of catenated circles can acquire negative supercoiling due to action of DNA gyrase. In supercoiled catenanes, the windings between catenated rings are concentrated in one region that is exposed to topoisomerases mediating DNA decatenation. C₁ Partially replicated DNA molecule forming an inter-sister knot. After completion of replicative strand separation, the crossings resulting from entanglements of such a knot get converted into crossings between two catenated rings. C₂. Partially replicated DNA molecule where a knot is formed within one freshly replicated sister chromatid. C₃. Upon completion of replicative strand separation the molecules with one intra-chromatid knot get converted into catenated rings, where one ring inherits the intra-chromatid knot. When the knot is removed by action of DNA topoisomerases before the decatenation, this results in formation of supercoiled catenated rings shown in panel (E).