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Chapter 14 Mechanisms of separation of the complementary strands of DNA during replication

A.I. ALEXANDROV, N.R. Cozzarelli, V.F. Holmes, A.B. Khodursky*, B.J. Peter, L. Postow, V. Rybenkov, and A.V. Vologodskii
Department of Molecular and Cell Biology, 401 Barker Hall, University of California, Berkeley CA 94720; *Stanford University Medical Center, Stanford CA 94305; Department of Chemistry, New York University, New York NY 10003

In this article, we will consider the separation of the complementary strands of DNA during DNA replication. We present a perspective on the field rather than an exhaustive review. Separation of the complementary strands of DNA, designated W and C, during replication is accomplished principally by the combined action of two types of enzymes - DNA helicases and DNA topoisomerases. The helicases are molecular motors that transduce the energy from the binding and hydrolysis of nucleoside triphosphates into breaking the H-bonds and stacking forces that hold the DNA duplex together [1]. The helicases are aided in this process by the helix destabilizing proteins which bind preferentially to single stranded DNA [2]. The topological constraint on all closed circular DNA and all linear DNA beyond a minimal size [3] requires that the complementary strands also be passed through each other during replication. This remarkable activity is the hallmark of the topoisomerases [4]. As we have discussed elsewhere, the concept of linking number (Lk) can be used to describe the process of DNA strand separation in any closed topological domain [5]. The Lk of DNA is one-half the number of crossings in plane projection of the W and C strands. A sign convention has been chosen so that the crossings of the W and C strands in ordinary B-DNA are positive. Lk is the sum of two terms: twist (Tw), or the local winding of the W and C strands around each other, and writhe (Wr), a measure of DNA 1

2 supercoiling [6]. The important consequence of topological constraint is that Lk remains unchanged by any deformation of the DNA backbone short of breakage and reunion. Thus, by unwinding DNA, helicases reduce Tw and thereby equivalently increase Wr. The reduction in Tw is stabilized by the binding of helix destabilizing proteins and ultimately DNA synthesis itself. The increase in Wr, however, is highly energetically unfavorable and cannot be readily stabilized. If not addressed, it would arrest replication fork movement. Topoisomerases come to the rescue and remove the excess Wr by reducing Lk. Thus helicases and topoisomerases work together in reducing Tw and Wr during replication. It is useful to consider not only the absolute value of Lk of a DNA but 0 also the difference from the Lk of a relaxed DNA, Lk . This difference is 0 0 designated Lk (Lk - Lk ). Supercoiling density (), Lk divided by Lk , is a length independent measure of the topological deviation of a DNA from 0 the lowest energy state. Lk is a topological invariant but Lk is not as its value depends on ambient conditions. When the helicases unwind DNA they 0 reduce Lk but cannot change Lk. Therefore, helicases generate a (+) Lk which is removed by the reduction in Lk by topoisomerases. Strand separation can be said to be complete only when the daughter duplexes are safely in daughter cells. Before then, the daughter DNAs could catenate and thereby reestablish an Lk between the W and C parental strands. Thus, chromosomal segregation and partitioning complete strand separation. We are concerned only with the Lk between the parental W and C strands as opposed to the Lk between the parental and daughter strands after semiconservative DNA synthesis. Until replication of a domain is complete and domain barriers are established, the daughter strands are interrupted and are therefore topologically irrelevant. DNA strand separation is a daunting task for the cell for a number of 2 -1 -1 reasons. First, unlinking must be very fast, about 10 sec fork in bacteria. But topoisomerases are slow. The turnover number of the relevant -1 topoisomerases is on the order of only sec [5, 7]. Therefore, a large number of topoisomerases are needed per fork. Instead of a sleek replication machine in which topoisomerases are in stoichiometric amounts with other replication proteins, we imagine a gaggle of topoisomerases breathlessly passing DNA through themselves as fast as they can. This requires not a little space along the DNA. The mechanical stress imposed by the (+) Lk generated by replication increases greatly as the length of available DNA diminishes below a minimal value [8], and the topoisomerases themselves require a long stretch of DNA [4] to reduce Lk. Thus, the corps of topoisomerases must act quickly and need a working space on the order of tens of kilobases per fork. Second, Lk must be reduced to exactly zero from a value of tens of millions for large eukaryotic chromosomes. What surveillance mechanisms


insure that all the local fluctuations in Lk exactly cancel? Even if topoisomerases somehow were able to reduce Lk to zero on average, strand separation would still not follow. This is because the product of DNA relaxation is a Gaussian distribution of topoisomers whose variance increases with DNA length [8]. Even for moderate-sized plasmids, let alone grand chromosomes, the great majority of molecules in a relaxed population have a non-zero Lk. A further complication is that strand separation requires the removal of all topological properties. Not only must Lk be zero, but all knots and catenanes must be absent as these too prevent separation of the DNA strands. Because of the difficulty in imagining how a global property like Lk could be nulled by locally acting enzymes, a topologist named William Pohl concluded that the strands of DNA could not be interwound as in the Watson and Crick model, but must instead be side-by-side [9]. Of course, Pohl was wrong, but his challenge to find the right explanation is contemporary. Third, Lk reduction must be coordinated with replication fork movement. If Lk gets too large, replication will stall. The cell cannot in advance build up substantial negative supercoiling as a prepayment on unlinking. Decreasing even modestly beyond the physiological value of -0.06 causes DNA to denature and promotes alternative secondary structures and RNA loops [8, 10], all with untoward effects. In the other direction, even a small increase in can turn off transcription or enzyme action in bacteria [11]. As a result, the value in bacteria is kept within narrow limits. Mutations in topoisomerases which upset that balance are lethal [12]. Fourth, many factors oppose unlinking. These include proteins which bind preferentially to duplex DNA over single stranded DNA. The very crowded confines of the cell augmented by the abundance of crowding agents also opposes unlinking on a mass action basis [13, 14]. A crude calculation suggests that the daughter bacterial chromosomes within a cell 4 would be catenated to each other perhaps 10 times at equilibrium. Moreover, each chromosome at equilibrium would be in the form of a fantastically complex knot with a similar number of crossings. Instead, the -5 frequency of chromosome missegregation is only on the order of 10 [15]. Thus, the number of links between bacterial chromosomes may be a billion times less than at equilibrium. The different topoisomerases in cells play complementary roles in unlinking DNA during replication. Type-1 topoisomerases break one strand of the DNA duplex and pass another segment of DNA through it, whereas type-2 enzymes break both strands of DNA and pass another segment of DNA through the transient double-stranded break [16]. The type-1 enzymes are divided into two classes. Type-1A enzymes bind single stranded regions or gaps preferentially, and can relax () supercoils but not (+) supercoils [4].

4 Therefore type-1A enzymes such as topo I and topo III in eubacteria and topo III in eukaryotes increase Lk and oppose unlinking. Although topo III can decatenate interrupted DNA [17] (and perhaps intact DNA in the presence of a helicase), genetic data prove that type-1A enzymes are not essential for replication [18]. On the other hand, type-1B enzymes, such as eukaryotic topo I, bind double-stranded DNA well and can relax either (+) or () supercoiled DNA [19]. Eukaryotic topo I can support replication elongation by itself [20]. The type-2 topoisomerases, DNA gyrase and topo IV in eubacteria, and topo II in eukaryotes, are essential for complete replication and segregation [21]. Given the challenges of DNA strand separation and its vital importance, it is not surprising that cells have developed many strategies for promoting unlinking. Some of these functions overlap so that their removal by mutations or drug treatment may not be lethal or, indeed, may only be apparent when parallel paths are removed. We will summarize seven different factors that contribute to strand separation. While additional features surely remain to be discovered, enough is known to define many of the critical issues.

1. Supercoiling promotes unlinking.

Negative supercoiling, by reducing Lk, puts a small down payment on strand separation. The major benefits, though, of negative supercoiling for strand segregation are conformational, not topological. By winding neighboring DNA segments around each other, supercoiling promotes a specific condensation of daughter DNAs upon themselves rather than a condensation of daughter DNAs with each other or with parental DNA. Computer simulations show that supercoiling increases the local concentration of DNA segments in the same molecule by more than two orders of magnitude [8]. Supercoiling greatly reduces the internal volume available for linking of plasmids or of domains in linear DNA. The equilibrium constant for catenation decreases about exponentially with supercoiling [22]. For a 7-kb plasmid, a of -0.06 reduced catenation by 400-fold. The magnitude of the enhancement of decatenation will increase with DNA length because the volume of supercoiled DNA grows linearly with length, whereas that of a coil increases as a power of length. Thus, we estimate that supercoiling of the E. coli chromosome will decrease the probability of catenation for each 50-kb domain by about 2,000 fold. Supercoiling also increases the rate of decatenation. Supercoiled catenanes comprised of 3.5 kb DNA circles were unlinked about 4-fold faster than relaxed catenanes [23].


These in vitro data are complemented well by the physiological results in vivo. In the absence of a functional DNA gyrase, dumb-bell shaped nucleoids interpreted to be catenated chromosomes accumulate inside the E. coli cell [24, 25]. Similarly, partitioning of plasmid DNA was dramatically affected in gyrase mutants [26, 27, 28]. Bacterial mutants in which chromosome partitioning is blocked are called par mutants [29]. Two early par isolates, parA and parD, encode the two subunits of DNA gyrase [30, 31]. These results were originally misinterpreted to mean that gyrase was the major decatenating enzyme in bacteria. After the discovery of topo IV [32, 33] and its function [34], catalysis of decatenation was reassigned to topo IV, but this unwittingly obscured the important role of DNA gyrase in chromosome segregation. This role has recently been established. Catenated plasmids were generated by recombination in cells in which was varied by mutations in gyrase and other topoisomerases. Decatenation was extremely efficient at normal values but a drop in supercoiling to a of -0.03 reduced decatenation to near background levels [11].

2. Unlinking takes place throughout a replicating domain by the complementary action of topoisomerases
Our view of the conformations of replication intermediates in bacteria and the roles of topoisomerases is diagrammed in Fig. 1. The (+) Lk generated early in replication causes (+) supercoils to be formed ahead of the replication fork. These are converted to () supercoils by DNA gyrase, and replication proceeds [35]. This scenario, however, cannot explain unlinking late in replication where only a short stretch of DNA is left ahead of the fork. This region is too small for many plectonemic (+) supercoils to accumulate and also too small to compete with the replicated DNA for gyrase binding. Champoux and Been [36] suggested that a rotation of the replication fork would allow the mechanical stress to be distributed into the replicated DNA, forming DNA crossovers called precatenanes [5]. Precatenanes are so named because they have an analogous structure to catenanes and are converted to catenane interlinks if not removed before the completion of replication. Precatenanes should be efficiently relaxed by topo IV, and thus topo IV would support replication elongation by removing links behind the replication fork. To summarize, very early in replication unlinking would be predominantly by gyrase ahead of the fork. Late in replication it would be carried out by topo IV behind the fork. At other times, the enzymes would cooperate to remove the (+) Lk distributed in precatenanes and supercoils. We have recently confirmed the Champoux and Been proposal. We found that superhelical stress is indeed distributed throughout the purified replication intermediates [37]. Data from both gel electrophoresis and

6 electron microscopy showed that partially replicated plasmids contain both precatenanes in the replicated region and supercoils in the unreplicated region. As the replicated region becomes larger, progressively more of the Lk is expressed as precatenanes. A complementary experiment also indicated that precatenanes are active substrates for unlinking. E. coli topoisomerase III supports elongation and complete decatenation of plasmid DNA in vitro [38]. Because topo III is a type-1A enzyme, it cannot remove (+) supercoils in front of the replication fork. The conclusion is that it removes (+) precatenanes behind the fork.

Figure 1. Scheme for unlinking DNA during bacterial DNA replication. The parallel lines represent the DNA double helix and the shaded rectangles are domain barriers. Replication is from left to right; () parental strands, (---) daughter strands. DNA replication generates (+) Lk. Early in replication most of the DNA within a domain is unreplicated and the (+) Lk is expressed as (+) supercoils ahead of the fork. By converting these (+) supercoils to () supercoils, gyrase reduces Lk. Late in replication, there is little unreplicated DNA and the (+) Lk from replication is expressed mostly as (+) precatenanes, which can be effectively removed by topo IV.

The two distinct bacterial type-2 topoisomerases are well adapted to their different roles in unlinking during replication. Gyrase efficiently converts


(+) supercoils to () supercoils, and thus is ideally suited to act in front of the replication fork. As a result, inhibition of gyrase with quinolones causes a relatively fast stop in replication [39]. On the other hand, topo IV is a potent decatenase which can efficiently remove precatenanes behind the replication fork. Drug inhibition of topo IV causes a slow stop in replication [40], consistent with its role behind the replication fork. When both enzymes are inhibited, replication is stopped faster than when either one is blocked [40]. This complementarity of gyrase and topo IV in replication elongation is analogous to their roles in the final decatenation and segregation of the replicated daughter chromosomes, where decatenation by topo IV is powerfully stimulated by the supercoiling of the DNA by gyrase. A similar cooperation between topoisomerases is present in eukaryotic cells. Topo I may operate predominantly ahead of the fork in place of DNA gyrase. DNA gyrase can barely knot or catenate duplex DNA and eukaryotic topo I, as a type-1 enzyme, cannot at all [42]. Thus, it may be that these enzymes are safer alternatives for unlinking during replication: even at the high concentrations required to support rapid elongation, they will not knot and catenate the daughter DNAs. Eukaryotic topo II should have the same role as topo IV in removing (+) precatenanes behind the fork. These powerful enzymes can knot and catenate DNA, but two factors limit the approach to topological equilibrium. First, type-2 topoisomerases actively remove links past the equilibrium position (see Section 4). Second, the type2 enzymes seem to be compartmentalized in cells [41] and can thus only act in the domain in which they reside. What happens very late in replication after DNA synthesis is complete provides an answer to Pohls challenge. Pohl is correct: organisms do not succeed in reducing Lk to 0 during the elongation phase of replication. As a result, their daughter chromosomes are wound around each other. These links are removed with high efficiency by the same type-2 topoisomerases that remove precatenanes, and unlinking is complete. This staged unlinking was first demonstrated with eukaryotic circular viral DNA by the finding that inhibition of topo II resulted in catenated products [43]. In eubacteria, inhibition of topo IV resulted in catenated plasmids [34]. Chromosomes suffer the same fate. Mutations in either of the two subunits of topo IV are par mutants [32, 33], and in topo II mutants of both S. cerevisiae and S. pombe, chromosome segregation was blocked [20, 44, 45].

3. Topological domains
The size and complexity of chromosomes demands organization, and the topological domain is a key unit of chromosomal organization. The ends of a domain are constrained so that they cannot rotate in relation to each other. The breaking up of huge chromosomes into 50-100 kb domains allows them to replicate essentially like a series of plasmids with manageable size and topology. The evidence for domains in E. coli chromosomes is indirect but, nonetheless, persuasive. Isolated nucleoids from E. coli are supercoiled and multiple nicks by DNase are required to relax the chromosome [46]. The topological domains are interpreted to be the feature of the chromosome that prevents a single nick from relaxing the whole chromosome. Treatment of nucleoids with protease or RNase caused the domains to disappear, suggesting that both proteins and RNA are necessary to maintain the domain boundaries. There is also physiological evidence for topological domains. Trimethylpsoralen binding to chromosomal DNA in vivo is enhanced by () supercoiling. Gamma ray nicking relaxes DNA and therefore reduces trimethylpsoralen binding. However, about 100 nicks were required per chromosome [47]. Finally, a domain structure of chromosomes has been suggested visually. E. coli nucleoids appear in electron microscopy as a rosette of about 100 supercoiled loops [48]. Each loop is a separate domain because occasional loops are relaxed but the adjacent ones are still supercoiled. Because the number of supercoiled loops is approximately equal to the number of nicks required to relax the E. coli chromosome, it has been suggested that the structural loops seen by microscopy and the topological domains are the same [49]. The organization of chromosomes into separate topological domains would have several important consequences for unlinking DNA during replication. First, if chromosomes were just a single huge domain, the difference in energy between a small Lk and one of zero would be negligible. Yet a non-zero Lk blocks partitioning. The problem is greatly ameliorated by the existence of domains. At any one time, perhaps only 50100 kb of DNA needs to be surveyed by topoisomerases, not hundreds of megabases. This simplification provides a partial solution to Pohls challenge but raises a new question: what mechanism ensures that domains once replicated are, and remain, link-free? A spatial separation of the domains after their replication is complete could accomplish this. Second, in the absence of domains, any interruption of DNA would destroy the supercoiling of the whole chromosome. The act of replication itself interrupts DNA as the growing points must be free (Fig 1). But as we discussed in Section 1, supercoiling is vital to chromosome segregation in


bacteria. Domains would seal off the interrupted section of DNA and prevent it from affecting the topology of the rest of the chromosome. Third, the domain barriers surrounding a replication fork prevent () supercoils in neighboring DNA from moving into the replicating domain to become () precatenane links (Fig 2). The removal of () precatenanes would increase Lk and thereby impede replication. Conversely, the precatenanes in the replicating domain are prevented by domain barriers from winding the daughter DNAs around each other and thereby impeding partitioning (Fig 2).

Figure 2. Domain barriers and replication in bacteria. The symbols are as in Fig 1. The typical replicating domain will have (+) supercoils ahead of the fork and (+) precatenanes behind it. Outside of this domain, both the replicated and prereplicated DNA will be () supercoiled due to the action of DNA gyrase. The domain barriers allow () supercoiling by sealing off interruptions from a neighboring domain. They also localize the topological structures involved in unlinking to the replicating domain and thereby prevent Lk from diffusing into the replicated DNA as precatenanes.

4. Active unlinking of DNA

We argued in Section 2 that complete unlinking is achieved in two steps. The links remaining in the first step, in which synthesis is completed, are removed in the second. But what insures the absolute efficiency of the second step? Crude estimates, based on extrapolating the data obtained in studies of equilibrium catenation in plasmids [22], predict that each supercoiled domain in the E. coli chromosome would have a probability of about 0.1 to be catenated. Although this number does not seem exceptionally 90 high, it means that only one in about 104 (one in 0.9 ) cells would have completely untangled chromosomes. An important factor is that type-2 topoisomerases that operate in the second step of unlinking are like Maxwellian demons that are not satisfied with bringing the number of links down to the equilibrium value but actively unlink DNA [49]. No thermodynamic law is violated in this reaction because

10 these topoisomerases unlink DNA at the expense of ATP hydrolysis. All forms of DNA topology supercoils, knots, and catenanes are actively removed by type-2 topoisomerases. However, consistent with their function to support chromosome replication and segregation, the enzymes work most efficiently in decatenation and unknotting [51]. Bacterial topoisomerase IV was found to reduce the equilibrium fraction of catenanes in a mixture of relaxed 10-kb plasmids by more than an order of magnitude [50]. It is not clear a priori how large the effect would be for longer and supercoiled molecules, which mimic the topological state of the chromosome.

5. Effective DNA concentration is less than global DNA concentration

The DNA inside of cells is at a very high concentration, up to 100 mg/ml [52]. The effect of this high concentration is exacerbated by the condensing and macromolecular crowding agents [13]. We have measured an effective concentration of DNA, a term similar to activity in chemistry. We define the effective concentration operationally as that concentration in vitro which gives the same amount as an intermolecular reaction in vivo. In E. coli we used the site-specific recombination mediated fusion of circular plasmids as the reporter intermolecular reaction and found that the effective concentration was about one-tenth of the global or chemical concentration [53]. The probability of catenation depends on DNA concentration [22]. We examined as a second measure of effective concentration in E. coli, the in vivo steady-state catenation across a wide range of plasmid DNA copy numbers. We obtained a semilogarithmic dependence of the steady-state plasmid catenation on the chemical plasmid DNA concentration [40]. We found that the activity of DNA, especially at higher concentrations, is more than 10 times lower than its chemical concentration.

6. Mechanical forces in unlinking: chromosome segregation

The last step in unlinking is chromosome segregation. Without separation, the chromosomes never become entirely unlinked, and cell division would result in cleavage or nondisjunction. Two major forces are responsible for separating chromosomes so that topoisomerases are able to complete their job of unlinking: the forces that actually pull chromosomes apart and those that condense chromosomes upon themselves. The spindles are the chief mitotic and meiotic forces that pull chromosomes apart in eukaryotic cells. As long as the rate of pulling by the



spindle fibers is slower than disentanglement by topoisomerases, separation of strands can be completed in anaphase. This separation will be irreversible. The critical question is one of timing. Are the links between the W and C strands removed during or before anaphase? The answer is that the vast majority are removed before, but a small but critical number are removed during anaphase (reviewed in [45]). Prokaryotes do not have an organized spindle, but recent data indicate a movement of daughter origins away from each other. In Bacillus subtilis, this is dependent on the SpoOJ-Soj system. SpoOJ binds to sites near the origin of replication, where it helps in establishing the proper orientation of the origin with respect to the cell poles [54]. In E. coli, Niki and Hiraga used in situ hybridization to follow the movements of the oriC and ter regions of the chromosome during the cell division cycle [55]. They found that the origin is localized to the cell pole, and that one copy of the origin migrates to the opposite cell pole after duplication. The terminus migrates to the midcell region where it duplicates shortly before cell division. These movements may be achieved by the action of the MukB protein. Hiraga proposed that MukB is a novel bacterial motor that binds to the chromosome and pulls apart the decatenated sister chromatids from their midcell position to the poles of the late predivisional cell [15]. mukB mutants of E. coli are defective in the correct partitioning of replicated chromosomes, which results in the appearance of anucleate cells during cell proliferation. However, the partitioning defects of mukB mutants can be partially suppressed by a mutation in topA, the gene for topo I, implying that an increase in the level of supercoiling, and thus condensation, can help overcome a defect in the active movement of the chromosomes. Interestingly, the system responsible for partitioning the bacterial chromosome is independent from that required for partitioning of low copy number plasmids, which duplicate in the mid-cell region and actively move to the 1/4 and 3/4 positions in the cell [56]. Condensation of chromosomes clearly plays a role in unlinking in eukaryotes and prokaryotes (reviewed in [57]). In eukaryotes, chromosome condensation occurs during metaphase, simplifying the process of disentangling during anaphase. Because the chromosomes take up less space, the process of separation of sisters has actually already begun. The clearest evidence of the molecular basis of mitotic chromosome condensation comes from the SMC (Structural Maintenance of Chromosomes) proteins (reviewed in [58]). SMC mutants in yeast have defects in the segregation of mitotic chromosomes [59, 60]. The condensins of Xenopus contain SMC proteins. These proteins are required for the assembly and structural maintenance of mitotic chromosomes in vitro [61].

12 In prokaryotes, a chromosome condensing system is formed by the products of the E. coli genes crcA, cspE and crcB. These were identified in a screen for mutations conferring resistance to camphor, a chemical known to induce chromosome decondensation [62]. CspE is a cold-shock-like protein that binds to several promoters and also to the corresponding mRNA. Overproduction of CspE partially suppresses the defects of a mukB deletion. The finding that this condensation protein binds RNA is intriguing because RNA has long been suspected to participate in holding the nucleoid together. The function of the other two proteins is unknown [57].

7. Recombination-promoted unlinking at the termination of replication

Circular DNAs face a special challenge during replication in that any odd number of homologous recombination events will fuse the daughter DNAs into a single dimeric molecule. Such dimerization would, of course, interfere with the equal partitioning of plasmids and chromosomes into the two new daughter cells [63, 64, 65]. Many circular genomes encode recombination activities that specifically resolve replication dimers into monomers. The topological challenge is to achieve the resolution without introducing catenane links. Because site-specific resolvases generally have catenated products [67], monomerization probably occurs before the second stage of unlinking. In E. coli, both the bacterial chromosome and plasmids such as ColE1 make use of the dif/Xer site specific recombination system [68, 69]. The Xer recombinase, a heterodimer of the XerC and XerD proteins, acts at the dif site located near the terminus of chromosome replication [70]. The dif/Xer system is broadly conserved. Homologues to XerC and XerD have been found in all bacteria tested and several also have sequences similar to dif [71, 72]. Mutants in XerC, XerD, or dif show a par phenotype with filaments of cells containing multicopy nucleoids that have not been properly partitioned [73]. This phenotype is not seen in dif recA double mutants in which crossovers leading to dimerizations cannot occur [73]. XerC and XerD are closely related members of the integrase family of enzymes, which includes the Cre recombinase important for phage P1 dimer resolution [74, 75]. The Xer proteins bind cooperatively to the dif site and related sequences in vitro. Mutations engineered into the defined chromosomal binding site alter recombination efficiency in vivo [76]. The mechanism by which the Xer heterodimer acts to assure segregation of monomers at the end of replication is not clear. The position of the dif site is important as dimer resolution is ablated by moving the dif site out of a small region near the terminus of replication [77]. Dimer resolution by Xer can be replaced by the lox/Cre system when a lox site is placed near the dif



site, but not by the Tn3 res/resolvase nor the cer/Xer systems [68]. One model consistent with these data is that after replication, but before partitioning, the recombinase acts repeatedly so that regardless of the starting state, the chromosome will be monomeric for some of the time when partitioning occurs [68]. This model offers one solution to the question of how a locally acting protein could resolve chromosomal dimers without sensing the number of crossovers in 4.7 megabases of DNA.

Acknowledgements Work described in this review was supported by grants from NIH, NSF, and NIEHS.

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