cromosomeStructure.pdf

Chromosome StructureN Patrick Higgins, University of Alabama, Birmingham, Alabama, USA

Genes are organized into discrete cellular structures called chromosomes that coordinate

DNA replication and distribution of replicated genetic copies between two daughter cells.

As vehicles of genetic transmission, chromosomes play a central role in Darwinian

evolution.

Abundance

Biology is divided into three great kingdoms: Eubacteria,Archaea and Eukaryota. Bacteria (Eubacteria and Ar-chaea) are ubiquitous in the environment, and these smallsingle-celled organisms growover an amazinglywide rangeof environmental conditions. For example, bacteria cangrow at temperatures below freezing, and certainmembersof the archaea can grow at depths of over threemiles and attemperatures exceeding 6008F and 200 atmospheres ofpressure. Eukaryotes, which include themore conspicuousfungi and all plants and animals, are usually recognized asthe predominant life forms on earth. They are subjects ofgreat biological interest and they dier from bacteria byhaving a larger cell size and by compartmentalizingchromosomal deoxyribonucleic acid (DNA) in a nucleus,which separates it from the protein-making cytoplasm.None the less, considering the large cell numbers and wideenvironmental growth ranges, bacteria easily account forthe majority of DNA biomass on earth.The chromosome is the heart of a central paradox in

evolution. How do species in the three kingdoms remainthe same over long periods of geological time and alsogenerate sucient variability to produce new species,sometimes relatively rapidly? Stability versus change is acrucial dichotomy in molecular biology. The events thatbring about stability and change in DNA structure involveprocesses of replication, transcription and recombination.Similar mechanisms operate in the three living kingdoms,but the key molecular mechanisms that control andcatalyse these events are understood best in a eubacterium:Escherichia coli.

Chromosome Size

Free-living bacteria need genetic information to synthesizeproteins for executing vital functions.Most bacteria have asingle chromosome with DNA that is about 2Mbp (megabase pairs) long (1Mbp5 1 000 000 base pairs), but theDNA content of dierent species varies from 0.58 togreater than 9Mbp of DNA, and some bacteria havemultiple chromosomes. For example, Leptospira has twochromosomes of 4.4 and 4.6Mbp and the largest bacterial

genome yet analysed is that of Myxococcus xanthus, with9.2Mbp (9 200 000 bp). However, the best studied organ-ism in nature is E. coli, which has a 4.6-Mbp chromosomewith 4288 genes for proteins, seven operons for ribosomalribonucleic acids (RNAs), and 86 genes for transferRNAs.The E. coli chromosome contains numerous gene

families, each family having evolved from a commonancestor. The largest gene family is comprised of 96 three-component (ABC) transporters, which are membrane-bound machines that import and export a variety of smallmolecules and proteins. By contrast, the smallest free-living organism is Mycoplasma genatalium with a 0.58-Mbp genome that encodes 468 protein genes, oneribosomal RNA operon and one ABC transporter. BothE. coli and M. genatalium have complete information forthe synthesis of cell walls, cell membranes and criticalenzymes of intermediary metabolism, plus the RNAmolecules, ribosomal proteins and a clutch of enzymes(the replisome) to replicate DNA eciently. Putativefunctions for about a quarter of the genes of E. coli remainto be discovered. A reasonable estimate is that 150200protein-encoding genes would be essential for a basicbacterial lifestyle (in a rich medium).Eukaryotic organisms generally have larger chromo-

somes than bacteria. For example, the yeast Saccharo-myces cerevisiae has about 6000 genes (50% more than E.coli), whereas mammalian cells contain 1000 times (perhaploid equivalent) the DNA of an E. coli cell. In humansthe 5000Mbp of haploid DNA is distributed among 22autosomes and two sex-specic chromosomes. EukaryoticDNA is localized in a compartment, the nucleus, which isseparated by a phospholipid-containing membrane fromcytoplasmic ribosomes and protein translation activity.During cell division, the eukaryotic nuclear membrane

Article Contents

Introductory article

. Abundance

. Chromosome Size

. Plasmids

. Chromosome Shape

. Enzymes of DNA Topology

. Chromatin

. DNA Replication

. Eukaryotic Segregation

. Transcription

. Recombination

. Chi Sites

. Transposons

. Site-specific Recombination

1ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net

breaks down once per cell cycle to distribute the 46 diploidchromosomes equally between two daughter cells.

Plasmids

In addition to the large chromosome, many (most?)bacteria have additional DNA molecules called plasmids(or episomes.) Plasmids are separate DNA molecules thatcontain a replication origin which allows them to multiplyindependently of the host chromosome. Plasmids range insize from 1kbp (Kilo base pair) (1000 bp) to 100 kbp, andthese DNA molecules encode genetic systems for specia-lized functions. Some plasmids make extracellular appen-dages that allow bacteria to infect and colonize sensitiveeukaryotic hosts. Plasmids often carry genes that confer onbacteria the ability to survive in the presence of antibioticssuch as tetracycline, kanamycin and penicillin. Manyplasmids also contain genes that promoteDNA transfer sothat plasmid genes can move into other bacterial species.Plasmid transfer has caused the emergence of bacterialpathogens that are resistant to most of the usefulantibiotics in medicine, with notable examples includingmultidrug-resistant strains of Staphylococcus and Myco-bacterium tuberculosis.In most eukaryotes, plasmids are rare. However, S.

cerevisiae contains a plasmid called the 2-m circle whicheciently partitions to new daughter cells at every celldivision. ThisDNA serves as a convenientmodule for genecloning and performing genetic experiments in yeast.

Chromosome Shape

On a macroscopic scale, bacterial chromosomes are eithercircular or linear. Circular chromosomes are mostcommon, at least among the best-studied bacteria. How-ever, the causative agent of Lyme disease, Borreliaburgdorphei, has a 2-Mb linear chromosome plus 12dierent linear plasmids. Eukaryotic chromosomes areinvariably linear, and they have two ends, each carrying aspecial structure called a telomere, and a organized regioncalled the centromere which allows the chromosome toattach to cellular machinery that moves it to the properplace during cell division.One critical facet of chromosome structure is that DNA

is a plectonemic helix, whichmeans that two helical strandsentwine about each other. For duplex DNA the twoantiparallel strands, often referred to as the Watson andCrick (red and blue in Figure 1), are interwound once forevery 10 bp. Because of this wound conguration, bio-chemical transactions that involve strand separationrequire chromosome movement (spin) about DNAs longaxis. The processes of DNA replication, recombinationand transcription all require DNA rotation, and during

DNA synthesis the rotation speed approaches 6000 rpm.Chromosomal DNA molecules are very long and thin, soDNA must fold many times to t within the connes of abacterial cell. How does DNA twisting transpire withoutchromosomes getting tangled up? The problem wasimportant enough that Watson and Crick considered aparanemic helix, which is a pair of coils that lay side by sidewithout interwinding. Strands of a paranemic helix canseparate without rotation (Figure 1). None the less DNA isplectonemic, and keeping chromosomes untangled re-quires a special class of enzymes called topoisomerases.

Plectonemic Paranemic

Eukaryotic chromatin Prokaryotic chromatin

Gyrase

Solenoidalsupercoils = 0.05

Histoneassembly

DeproteinateH-NSHU

Interwoundsupercoils = 0.05

TopoIV

TopoII

(a)

(b)

5kbpPlasmid

Figure 1 (a) DNA is a plectonemic helix (left) rather than a paranemichelix (right), and thus it must spin axially to undergo replication,transcription and extended pairing for homologous recombination.Supercoiling in circular bacterial chromosomes is maintained by theconcerted action of DNA gyrase, which introduces negative supercoils atthe expense of adenosine triphosphate (ATP) binding and hydrolysis, andTopoI plus TopoII, which remove excess negative supercoils. Negativesupercoiled DNA adopts an interwound conformation. However,solenoidal supercoils can be stabilized when DNA is wrapped on a proteinsurface. This is the mechanism of supercoil formation in mammalian cells(centre left). Most bacteria have nonspecific DNA-binding proteins such asHU and H-NS, which stabilize supercoils but are much less effective atcondensing DNA compared with true histones (centre right). (b) Bacteriaand eukaryotes both have an enzyme, TopoIV and TopoII respectively,designed to unknot and untangle DNA.

Chromosome Structure

2 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net

Enzymes of DNA Topology

The enzymes that solve the untangling problem aretopoisomerases. Topoisomerases break and rejoin DNAmolecules, thereby allowing individual strands to pass onethrough another. The number of phosphodiester bondsbroken and reformed per reaction cycle divides topoi-somerases into two classes. Type I enzymes, which includeTopoI and TopoIII (the odd-numbered topoisomerases),break one strand per cycle, and type II enzymes (evennumbered), gyrase, eukaryotic TopoII, and E. coliTopoIV, break two strands simultaneously. These en-zymes have two important roles: they provide a swivel toallow processes such as replication and transcription toproceed unimpeded, and they untangle knots and inter-chromosome links between DNA molecules.In all organisms DNA becomes organized into super-

coils, which are turns of the double strand over theinterwound twists of the WatsonCrick helix. Supercoilscan be either positive or negative; negative supercoils areopposite to the handedness of the WatsonCrick turns,and positive supercoils have the same handedness. Super-coil density is dened by a term s, which represents thenumber of superhelical turns divided by theWatsonCrickturns of a double helix. Supercoiling inuences theWatsonCrick structure and, like the spring, the freeenergy of supernegative supercoils increases exponentiallywith quantity. Enzymes that unwind DNA to carry outtheir function (RNA polymerase and DNA replisomes)may sense and utilize supercoiling energy. However, oncenegative supercoiling has reached a critical density, theWatsonCrick structure will unwind locally, forcingDNAinto alternative structures to relieve tension. Well-studiedsupercoil-dependent alternatives to the WatsonCrickform of DNA include left-handed Z-DNA, cruciforms,and triple-stranded or H-form DNA.In eubacteria, gyrase, TopoI and TopoIV maintain in

vivo supercoiling levels, s5 2 0.05 to 2 0.075. Gyrase isunique for its ability to introduce negative supercoils intorelaxed, positively or negatively supercoiled DNA at theexpense of adenosine triphosphate (ATP) binding andhydrolysis. An essential enzyme in bacteria, gyrase iscritical for DNA reactions that include recombination,replication, transcription and chromosome segregation. Invitro, DNA gyrase can supercoil DNA to a value ofs5 2 0.1, a level at which many sequences adopt analternative structure. In vivo, s is buered by the counter-active relaxing activity of TopoI and TopoIV. The secondcritical function of topoisomerases is untangling andunknotting DNA (Figure 1b). Eukaryotic TopoII and E.coli TopoIV eciently unknot and untangle chromo-somes, and after replication this activity allows thechromosomes to segregate into daughter cells.Although an average eukaryotic nucleus is larger than

an E. coli cell, nuclear DNA is even more concentratedthan bacterial DNA. Rather than being supercoiled by an

enzyme such as gyrase, eukaryoticDNA is wrapped tightlyaround nucleosomes, generating solenoidal supercoils thatcondense DNA 8-fold (Figure 1). A nucleosome containsfour subunits: histones H2A, H2B, H3 and H4. If histonesare stripped fromDNAbyprotein denaturants, eukaryoticDNA adopts the interwound structure of bacterialchromosomes, with about the same negative superhelixdensity (Figure 1).Eukaryotes have three topoisomerases: TopoI and

TopoIII are type 1 enzymes that break only one strand,and TopoII is the only type 2 activity that breaks bothstrands simultaneously. Eukaryotic TopoI removes bothpositive and negative supercoils from DNA, whereasTopoII carries out unknotting and decatenating reactions(see below). TopoI and TopoII are essential for eukaryoticcell viability; the functions of TopoIII in both eukaryotesand bacteria remain undened.

Chromatin

Chromatin is a term that refers not just to DNA but to theproteins attached to a chromosome. In the dimensions ofB-form DNA, E. coli is a sphere that is 6 kbp long (8 nm)and 4 kbp wide (2 nm), so the 4.6-Mbp chromosome mustbe folded many times to t within a cell. Negativesupercoiling forces DNA into an interwound congura-tion (Figure 1). Interwound supercoiling is produced at theexpense of ATP by the enzyme gyrase, and supercoilingproduces two important consequences. First, the DNAmolecule doubles back on itself so that length is halvedrelative to that of the linear form. Second, supercoiledbranches are dynamic so that opposing DNA sites in theinterwound network are constantly changing. A proteinbound to DNA in one supercoiling domain interacts morethan 100 times more frequently with other proteins in thesame domain than it does with proteins bound to adierent domain. When DNA is liberated from cells bybreaking the peptidoglycan coat, chromosomes formbundled loops that represent domains. Such preparations(called nucleoids) behave as discrete bodies.Many reactions of the chromosome require the forma-

tion of intricate DNAprotein machines to replicate,transcribe or recombine DNA at specic sequences. Agroup of chromosome-associated proteins assists in theformation of these complexes by shaping DNA. Theseproteins are sometimes described as histone-like, althoughthey share no structural similarity with the eukaryotichistones (Figure 1). Chromosome-associated proteinsinclude HU, H-NS, intregation host factor (IHF) andfactor for inversion stimulation (FIS).HU is encoded by two genes, hupA and hupB, which are

closely related to each other and to the genes encoding thetwo IHF subunits. HU is the most abundant double-stranded DNA-binding protein in E. coli, although it also



binds RNA. Although HU shows a preference forstructurally bendable sequences, it binds DNA relativelynonspecically, forming complexes that stabilize negativesupercoils in double-strandedDNA(Figure1). InphageMutransposition reactions, HU binds to a exed DNAstructure called a transpososome with high anity andspecicity.H-NS is a second relatively nonspecic DNA-binding

protein that forms dimers, tetramers and possibly higher-order associations when bound to DNA. Like HU, H-NSconstrains negative supercoils. H-NS binds bendableDNA, which include AT-rich sequences often found nearbacterial promoters; H-NS also modulates the transcrip-tion of numerous operons in E. coli and of lysogenicphages.FIS protein forms homodimers and binds DNA at

specic sites. FIS was discovered nearly simultaneously intwo related site-specic recombination systems: the Hininversion reaction of Salmonella typhimurium and the Gininversion reaction of phageMu. FIS concentration rises inearly log phase when the protein has regulatory roles inreplication initiation at oriC and transcription of riboso-mal RNA operons, and then FIS abundance declines instationary phase.IHF protein is composed of two subunits (encoded by

the himA and hip genes) that are about 100 amino acidslong and which make stable heterodimers. IHF proteinbinds to a consensus sequence that was rst discovered inphage l, where it stimulates insertional and excisionalrecombination in vitro by a factor of 104. IHF bends DNAseverely (more than 908); such bends are critical for site-specic recombination in l, for repression and activationof phageMu, and for repressionactivation in a number ofcellular operons, including ilv, oxyR and nifA.In the eukaryotic nucleus, enzymes such as RNA

polymerase gain access to DNA, which remains histonebound throughout most biochemical transactions. Theway in which multiple proteins interact with nucleosome-coated DNA is not completely understood; however,protein access to DNA is modulated by histone structure.Histone phosphorylation and acetylation change chroma-tin structure. Genes that are transcribed contain nucleo-somes with acetylated subunits of histones H3 and H4,whereas inactive genes are bound by histones lackingactetyl groups. In addition to the histones, eukaryoticchromosomes contain regulatory proteins that are muchless abundant. One class of proteins, the high mobilitygroup (HMG) family of DNA-binding proteins, bendsDNA much like bacterial proteins IHF and FIS.

DNA Replication

The central problem in chromosome replication isgenerating two high-delity DNA copies and distributing

them precisely to compartments that become the daughtercells. To control chromosomal replication temporally, andto integrateDNAmetabolismwith other aspects of the cellgrowth such as membrane synthesis and cell wall expan-sion, E. coli makes a master regulator, the DnaA protein,which controls the onset of replication at a site called oriC.The bacterial replication cycle can be described by

progression through four stages. Less detail is available foreukaryotes, but many points are similar. As cells grow, themass of protein and membrane increases to a critical pointthat triggers the initiation of replication. Two replisomesare associated in a factory that moves to the cell centreduring chromosomal elongation. As DNA strands arepulled to the centre, replicated sister chromosomesmigratetoward opposite cell poles. Completion of DNA synthesisoccurs as the DNA that is pulled into the factory reachesthe terminus. At this point, chromosomes are tangledtogether (catenated), and all physical connections must beremoved to allow nal separation. When physical separa-tion is complete, cell wall synthesis forms two daughtercells.

Initiation

DNA replication starts at oriC near position 2 85 of thestandard E. coli genetic map (Figure 2). Initiation involvesthe DnaA protein-assisted assembly of two replisomes,each dedicated to replicate half a chromosome. Replisomecomponents include: the DnaB helicase, which is an ATP-dependent ring that moves along a single strand breakingopen the double helix for polymerase access; a dimericDNApolymerasemolecule with accessory cofactors; and acomplex called the primosome that triggers RNA-primedinitiation of new DNA strands on one side of the growingfork. Each replisome has a pair of polymerase subunits:one synthesizes DNA continuously along the strand with53 polarity, and the other subunit synthesizes DNAdiscontinuously in small fragments called Okazaki pieces.Thus DNA replication is semidiscontinuous.In eukaryotic replication, initiation steps are not as fully

understood as they are in E. coli. Initiation occurs at arssites, so called because they are autonomously replicatingsequences (Figure 3). Initiation is controlled by a group ofproteins called ORC (origin replication complex). Typi-cally, a chromosome has many ORC-binding sites, andbidirectional semidiscontinuous forks move out fromseveral ars sites on each chromosome until they convergewith other forks. Eukaryotic replication also occurs infactories, and most of the chromosome is replicated in asemiconservative and semidiscontinuous mode, as in E.coli. The eukaryotic replication fork behaves very similarlyto that found in E. coli.



Elongation

After initiation, duplication of the entire 4.6-Mbp E. colichromosome takes 40min. One replisome copies DNA inthe clockwise direction (replichore I), while the otherreplisome copies DNA counterclockwise (replichore II)(Figure 2). DNA polymerase synthesizes new strands at astaggering rate of 300 nucleotides per second. Replicationis semiconservative, meaning that one newly synthesizedstrand is made to match each parental template. BecauseDNA polymerases synthesize DNA only in the 5 to 3direction, and because replication forks copy bothtemplates at nearly the same time, there is asymmetry inthe mechanism of chain growth. One strand is made as asingle piece while the other strand is synthesized in a seriesof 1-kb segments, with each segment being initiated with asmall RNAmolecule. These are called Okazaki pieces, andthey are stitched together after removal of the RNAprimers. This semidiscontinuous pattern of DNA replica-tion is a recurring theme in organisms as diverse as plantsand humans.

Because replication is semidiscontinuous, the sisterchromosomes (shown at the bottom of Figure 2) arereplication isomers. The template strand with 5 to 3polarity in the clockwise direction (the Watson strandtemplate in Figure 2) always contains DNA replichore I,which is made discontinuously, and DNA replichore II,which is made continuously; the Crick strand templatealways has the opposite style. Mutation and recombina-tion rates dier between discontinuous and continuousreplication modes, so the sisters are not always equivalent.As replication proceeds, positive supercoils build up in

front of the fork, and the daughter chromosomes becomeentangled behind the fork (Figure 4). These two topologicalproblems are solved in similar ways in the two kingdoms.ForE. coli, DNAgyrase removes positive supercoils aheadof the fork, and TopoIV removes the links between thedaughter chromosomes, which are called catenanes.Eukaryotic TopoI eliminates the positive supercoilsformed ahead of the fork, and TopoII decatenates thechromosomes for segregation (Figure 4).

oriC

Replichore II

E. coliWatson strand template

Replichore I

terE

oriC

Replichore II

E. coliCrick strand template

Replichore I

dif

terDterA

terCterB

terF

Escherichia coli replication

ReplisomeDiscontinuous strand

Continuous strand

Semiconservative andsemidiscontinuous replication

(a)

(b)

Figure 2 (a) A replication fork is shown with a replisome as a blue box, moving and synthesizing DNA from left to right. Replication is semiconservative,meaning that a new strand is synthesized on each of the two parental template strands, and semidiscontinuous, meaning that the strand that followsthe fork with 5 to 3 polarity is made as one continuous piece while the strand that follows with overall 3 to 5 polarity is made discontinuously as Okazakipieces. (b) The two circular chromosomes show the products of bidirectional replication. Initiation starts at oriC (285) and proceeds clockwise in thezone called replichore I and counterclockwise through the zone called replichore II. Replication forks meet at a terminus near the dif site (234). Strandsmade in the continuous mode are shown in red, and discontinuous strands in blue. Also included in the map are the seven ribosomal RNA operons(arrowheads), five of which reside in replichore I and two of which are found in replichore II, six ter sites and the dif site. Note that the two daughters aresynthesized with different semidiscontinuous styles in replichores II and I so that the Watson and Crick strands are isomers.



One segment of a eukaryotic chromosome that isdierent from prokaryotic chromosomes is the tip of thechromosome, the telomere (Figure 3). Telomeres are

replicated by a special DNApolymerase called telomerase,which is related to the reverse transcriptase of retroviruses.Telomerase synthesizes a simple repeat sequence that isadded on to every chromosome using an RNA templatethat is part of the enzyme. In most organisms telomerase isnot expressed after cell dierentiation, and consequentlythe telomere sequences shorten with age, eventuallycausing cell senescence and death.

Termination

In E. coli, replication is completed in a region of thechromosome called the terminus, which is 1808 around thecircular geneticmap from oriC. Two special sites are foundnear the terminus. First, the terminus is surrounded by atleast six ter sites (Figure 2, blue boxes) which bind the Tusprotein. A terTus complex induces replication forks tostop or pause in a unidirectional fashion by impeding themovement of DnaB helicase. Ter sites work in only oneorientation, and they are ordered so that replication forkscan proceed to the terminus but not continue in thedirection opposite to normal fork movement. Thus, tersites ensure that replication produces no more than oneround of synthesis per initiation event. The second specialsite at the terminus is called dif, which functions tomonomerize dimeric chromosomes during segregation (seebelow in site-specic recombination). The processesinvolved in terminating eukaryotic replication forks arenot yet known.

Segregation

At the conclusion of replication of a circular chromosome,all physical barriers must be eliminated before daughterchromosomes completely separate. Replication leavessister chromosomes interlocked bymultiple catenane links(Figure 1). To uncu the DNA molecules, both gyrase andDNA topoisomerase work together. Once chromosomes

Watson

Telomere

Crick

Ars2

Centromere

Ars1

Telomere

Figure 3 Structure and replication pattern of a eukaryotic chromosome.Eukaryotic chromosomes are linear structures which have special structuresat each end called telomeres (green) and an organizer centre called thecentromere which attaches the chromosome to the spindle duringchromosome segregation. Replication is initiated at ars sites, andreplication is carried out semidiscontinuously so that the two strands arereplication isomers.

TopoIV Escherichia coli Gyrase

TopoII TopoIEukaryotes

+ Catenanes

+ Supercoils

Figure 4 Topology of DNA replication. Movement of a replication forkproduces positive supercoiling ahead of the fork and results inentanglements of the sister chromosomes, called catenanes, behind thefork. Positive supercoils are removed by gyrase in bacteria and by TopoI ineukaryotes, whereas TopoIV resolves catenanes in bacteria and TopoII ineukaryotes.



separate, cell wall synthesis can form a permanent barrierbetween daughter cells.Bacteria can constrict the cell cycle to allow doubling

times that are shorter than the 40-min period necessary tomake one complete chromosomal copy. Under conditionsof rapid growth, initiation at oriC speeds up so thatdaughter cells inherit one fully replicated chromosomeplustwo or more partially replicated chromosomes at the timeof cell division (dichotomous replication).

Eukaryotic Segregation

FollowingDNA replication in eukaryotes, which occurs ina part of the growth cycle called the S phase, cells movethrough a mechanical cycle called mitosis to distribute thereplicated chromosomes to each daughter cell (Figure 5).Mitosis proceeds through four stages. The rst is theprophase in which, after replication, each chromosomebecomes condensed. Stage 2 is metaphase, where two

changes occur: eachpair of replicated chromosomesmovesto the cell centre and then the nuclear membrane begins todissolve. In stage 3, anaphase, one centromere of each pairof chromosomes is attached to a set of bres called thespindle, and molecular motors pull one of each chromo-some pair to opposite cell poles. In stage 4, telophase, thenuclear membrane is reformed and daughter cells areseparated by the synthesis of a new septum.

Transcription

A fundamental problem in biology is understanding howorganisms respond to the wide variety of environmentalconditions that a cell or a population of cells encounters.As noted above, dierent bacterial species grow over anamazing range of temperatures and environmental niches,but even a single organism can adapt to widely varyinggrowth states, from aerobic growth on rich nutrients toanaerobic growth in minimal salts and a single carbon and

S phase Metaphase

Prophase

AnaphaseTelophase

Figure 5 Chromosome segregation in eukaryotes is completed in the mitotic cycle. Mitosis proceeds through four stages, prophase, metaphase,anaphase and telophase, as described in the text.



nitrogen source. One key to ecient growth is control ofgene expression at the level of RNA transcription. Theprocess of making of an RNA complement to aDNA geneis called transcription.To accommodate complex regulatory scenarios, chro-

mosomes are divided into discrete transcriptional modulescalledoperons.Anoperonhas two functional components:the operator (or control region) and the coding sequence,which encodes the protein or RNA product. Althoughearly studies focused on operons such as lac, trp and his,which produce several proteins from a single operator,73% of the 2600 dierent E. coli operons encode only oneprotein. The operator includes at least one binding site forRNA polymerase, which is the enzyme that makes acomplementaryRNAcopy ofDNA.RNApolymerase hasa core subunit, plus a specicity factor called sigma (s).Sigma factors direct RNA polymerase to bind a specicsubset of promoters. InE. coli there are four sigma factors:s70, which controls many housekeeping genes and mostintermediary metabolism operons;sE, which is specic fornitrogen xation genes; sS, which controls genes that areturned on in stationary phase; andiA, which is specic foragellar genes.In addition to RNA polymerase, promoters contain

both positive and negative regulatory proteins. Negativeregulatory proteins are called repressors, and they blocktranscription by either impeding access of RNA polymer-ase or by inhibiting its ability to initiate RNA chains. TheLacI repressor blocks expression of the lactose genes whenno lactose is available for metabolism. There are alsopositively actingproteins called activators,which stimulateRNA polymerase transcription, either by stabilizing itsbinding to a promoter or by stimulating the ability ofRNApolymerase to initiate RNA chains. One example of a well-characterized activator protein is the cyclic adenosinemonophosphate (cAMP)-binding protein (CAP), whichbinds to a large number of operator sites and stimulatestranscription when cAMP levels are high.In bacteria, replication and transcription can occur

simultaneously. Replication requires all attached, doublestrand-specic DNA-binding proteins to be temporarilydisplaced during complementary strand unwinding andsynthesis. The proteins that have to be removed include thechromosome-associated proteins (see section on Chroma-tin above), all repressors and operon activator proteins,and the RNA polymerase. Transcription presents a specialproblem for the replisome. Because transcription andreplication occur simultaneously, two situations arisewhere transcription machinery and replication machinerycollide. One case is where transcription and replicationmove in the same direction. Because a replisome pulls inDNA at 500 bp per second and RNA polymerasetranscribes DNA at 50 bp per second, replication forksrapidly overtake RNA polymerase, even when bothenzymes are headed in the same direction. The morestriking encounter occurs when DNA replication meets a

transcribing RNA polymerase molecule moving oppositeto the direction of replication fork movement. In bothcases, the replisomes can pass RNA polymerases withoutcausing them to release transcripts or dissociate fromDNA. However, most highly transcribed genes aretranscribed in the same direction as replication forksmove. This includes all seven of the ribosomal RNAoperons (black arrowheads in Figure 2b) and 53 of the 86transfer RNA genes.In eukaryotic chromosomes, the RNA polymerase

responsible for transcribing most genes is remarkablysimilar at the structural level to the E. coli RNApolymerase. However, regulatory mechanisms are dier-ent. Eukaryotic genes have a region called the promoterwhich is where RNA polymerase binds and startstranscription. However, polymerase binding and its abilityto initiate transcription is inuenced by sites calledtranscription enhancers that can be upstream or down-stream of the promoter (relative to the direction oftranscription). Enhancers act over very large distances,and so DNA looping is required to bring enhancers intocontactwithRNApolymerase at promoters. In addition toenhancers, there are proteins called co-activators thatmustbind toRNApolymerase to stimulate transcription.Whilethere are many questions about how co-activators work,one important known function is to modify histones byacetylating the H3 and H4 subunits.

Recombination

Chromosomes undergo three types of genetic recombina-tion: homologous recombination, site-specic recombina-tion and transposition. Homologous recombinationinvolves exchanges between regions of identical or nearlyidentical sequences that are 300 bp or longer, with theeciency of recombination increasing up to several kbp.Several repetitive sequences, including the seven copies ofthe ribosomal RNAoperon in theE. coli genome, are largeenough to stimulate ecient recombination (Figure 2b). InE. coli, the pathway of homologous recombination iscarried out and regulated by four genes: recA, recB, recCand recD. Homologous recombination is responsible forthe introduction of new information into the bacterialgenome through mechanisms of transformation, conjuga-tion and transduction. However, another importantfunction of recombination is DNA repair. Recombinationallows replication forks that have stalled or fallen apart torestart DNA synthesis, in addition to allowing onedamaged chromosome to be repaired using informationfrom a sister chromosome. Thus, in addition to being anagent of change, recombination helps chromosomesremain the same.Homologous recombination is critical for repair of

DNA damage caused by chemical modication by



alkylating agents, by ultraviolet light, and X-ray-inducedchromosome breaks. DNA damage is also caused byinternal oxygenmetabolism, which generates free radicals.Replication errors also causeDNAbreaks. TheRecABCDpathway corrects a damaged copy of a chromosome byusing identical sequences in a sister chromosome in thesame cell. As noted above,when rapid growth is underway,cells have more than one chromosome. RecA proteinserves two roles in repairing chromosome damage. First, itbinds to single-stranded regions of a broken chromosomeand facilitates a search for the homologous sequence, andthen forms a hybrid molecule that starts recombination.RecA also regulates gene expression. When DNA damageoccurs, RecA protein inactivates a repressor called LexA,which results in the expression of over a dozenDNA repairproteins. After DNA repair is complete, RecA proteinstops inactivating LexA repressor and DNA metabolismreturns to normal.Recombination is a critical repair pathway in mamma-

lian chromosomes as well. Proteins that carry outbiochemical reactions similar to the E. coli RecABCsystem have been identied. A protein called p53coordinates the activity of many DNA repair proteins.Repair enzymes are stored at chromosome telomeres, andafter a signal from p53 these proteins migrate to sites ofDNAdamage to restore chromosome function.Mutationsin DNA repair genes have been discovered to beresponsible for several human genetic syndromes thatresult in premature ageing and high spontaneous rates ofcancer.

Chi Sites

One sequence in E. coli inuences the eciency anddirection of recombination: the chi sequence,GCTGGTGG. A chi site stimulates recombination direc-tionally, by triggering the pairing of chromosome se-quences near chi. Like ter sites in replication, chi sites act inonly one direction. About 75% of all chi sites are arrangedin the clockwise direction in replichore I and counter-clockwise in replichore II, so that they instruct recombina-tion to proceed according to the direction of DNAreplication.

Transposons

Transposons are discrete genetic elements that move fromone site or from one chromosome to another, often withlittle regard to any specic DNA target sequence.Transposons sometimes carry with them genes forresistance to antibiotics such as tetracycline (Tn10) andkanamycin (Tn5). A transposon generally has invertedrepeats at the ends of the element, and encodes at least one

protein called the transposase, which binds the ends andstimulates transposon movement. The chromosome of E.coli has 42 dierent transposons that have invaded byhorizontal transfer from other bacteria or viruses.In eukaryotes, transposons (usually called retrotran-

sposons because of their similarity to retroviruses and theirdependence on reverse transcriptase for replication) makeup a large fraction of total chromosomal DNA. In humancells, sequences called short interspersed nuclear elements(SINEs), which are about 300 bp long are present in about106 copies and represent 5% of the mass of DNA in ahaploid genome. One particular SINE, called AluI, ispresent on average once every 5000 bp in every humanchromosome. There are also long interspersed nuclearelements (LINEs) of about 6 kb that are present in about105 copies and represent 15% of the haploid chromosomalmass. What function, if any, these sequences provide forthe host organism is questionable, but many geneticmutations have been attributed to gene disruption causedby recent transposon insertions.

Site-specific Recombination

Site-specic recombination provides an ecient mechan-ism for rearrangingDNA at sites less than 100 bp in length(below the level required for homologous recombination).Site-specic recombination systems cause insertion andexcision of dierent lysogenic viruses, inversion of regionsanked by inverted sites, and deletions when two sites aredirectly repeated in a chromosome. Bacteriophage lcontains the best-studied site-specic recombination sys-tem the Int/Xis system which allows a prophage tointegrate into the bacterial chromosome at one point calledattB, or subsequently to excise and replicate in the lyticmode. Site-specic systems use a protein recombinase tobind and recombine short sequences, usually about 20 bplong.One site-specic recombination system in E. coli plays a

crucial role in chromosome segregation. DNA synthesismay generate breaks (often on the discontinuous strand)that stimulate recombination between daughter chromo-somes. If an odd number of crossovers occurs betweendaughters, the chromosomes will be dimerized at thecompletion of DNA replication. In E. coli, about 15% ofthe normal replication cycles produce dimeric chromo-somes.The site-specic recombination system that resolvesthese molecules is composed of the dif site at the terminus(Figure 2) and recombination proteins XerC and XerD,which eciently separate daughter chromosomes justbefore cell division.In eukaryotes, site-specic recombination also hasmany

critical roles in biological development. One spectacularexample is themammalian immune system,where dierentchromosomal segments called V,D and J genes are cut and



spliced together to produce antibodies. The mammalianimmune system can make antibodies to a diverse array ofdierent structures by cutting andpasting genes together inan immense number of dierent ways. The antibodysystem is essential for life, because it protects an organismfrom invasion by bacterial and viral pathogens. Thus, site-specic recombination has evolved as one key mechanismthat changes the genome in a small subset of dierentiatedcells, and this ultimately allows an organism to survive andstay the same.With multiple transposons, ecient homologous and

site-specic recombination systems, the occurrence offrequent chromosome breaks, and the presence ofmultiplelong inverted and direct repeats, it would seem impossiblefor chromosomes to remain constant through time.Surprisingly, the genetic maps of E. coli and S. typhimur-ium are very similar after 140 million years of separationfrom a common ancestor. Thus, the eciency of enzyme

systems designed to promote chromosome stability isnearly equal to the forces that promote change.

Further Reading

Casjens S (1998) Bacterial genome structure. Annual Review of Genetics

32: 339377.

Charlebois RL (ed) (1999) Organization of the Prokaryotic Genome.

Washington DC: ASM Press.

Cozzarelli NR and Wang JC (1990) DNA Topology and its Biological

Eects. Cold Spring Harbor, New York: Cold Spring Harbor Press.

Kornberg A and Baker T (1991)DNA Replication, 2nd edn. New York:

WH Freeman.

Kuzminov A (1999) Recombinational repair of DNA damage in

Escherichia coli and bacteriophage l. Microbiology and MolecularBiology Reviews 63: 751813.

Neidhardt FC (ed.) (1996) Escherichia coli and Salmonella. Washington

DC: ASM Press.



Documents

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