A.I. Alexandrov, N.R. Cozzarelli, V.F. Holmes, A.B. Khodursky, B.J. Peter, L. Postow, V. Rybenkov...

8/3/2019 A.I. Alexandrov, N.R. Cozzarelli, V.F. Holmes, A.B. Khodursky, B.J. Peter, L. Postow, V. Rybenkov and A.V. Vologods

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Chapter 14

Mechanisms of separation of the complementarystrands 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. VologodskiiDepartment 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 eachother 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


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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 Wrby 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

also the difference from the Lk of a relaxed DNA, Lk0. This difference is

designated Lk (Lk - Lk0). Supercoiling density (), Lk divided by Lk

0, is

a length independent measure of the topological deviation of a DNA from

the lowest energy state. Lk is a topological invariant but Lk0

is not as its

value depends on ambient conditions. When the helicases unwind DNA they

reduce Lk0

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

reasons. First, unlinking must be very fast, about 102

sec-1fork

-1in bacteria.

But topoisomerases are slow. The turnover number of the relevant

topoisomerases is on the order of only sec-1

[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 replicationproteins, 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


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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 allknots 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.

IfLk 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

would be catenated to each other perhaps 104

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

frequency of chromosome missegregation is only on the order of 10-5

[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].


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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 topoIV 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 forcatenation 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

theE. 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].


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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 theE.

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 parmutants [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 themajor 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 thecomplementary 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 tocatenane 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


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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 itremoves (+) 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


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(+) 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 isanalogous 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 type-

2 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]. Chromosomessuffer 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].


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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 theE. 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 50-100 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


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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 impedingpartitioning (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 thesecond 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

high, it means that only one in about 104

(one in 0.990

) 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


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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 supercoiledmolecules, which mimic the topological state of the chromosome.

5. Effective DNA concentration is less than global DNAconcentration

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


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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]. InE. coli, Niki and Hiraga used

in situ hybridization to follow the movements of the oriCand terregions 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 mid-

cell 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

ofE. 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 ofdisentangling 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].


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In prokaryotes, a chromosome condensing system is formed by the

products of the E. coli genes crcA, cspEand 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 ofreplication

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 difsite 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 difsite 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 difsite

is important as dimer resolution is ablated by moving the difsite 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


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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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