9 Геном, плазмиды, вирусы (1160078), страница 6
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The branch "migrates" when abase pair to one of the two complementary strandsis broken and replaced with a base pair to the second strand. In the absence of an enzyme to directit, this process can move the branch spontaneouslyin either direction.(b)An important contribution to understanding homologous recombination is a model proposed by Robin Holliday in 1964, a version ofwhich is presented in Figure 24-28. There are four key features of thismodel: (1) homologous DNAs are aligned by an unspecified mechanism;(2) one strand of each DNA is broken and joined to the other to form acrossover structure called a Holliday intermediate; (3) the region inwhich strands from different DNA molecules are paired, called heteroduplex DNA, is extended by branch migration (Fig.
24-29); and(4) two strands of the Holliday intermediate are cleaved and thebreaks are repaired to form recombinant products. Homologous recombination can vary in many details from one species to another, butmost of these steps are generally present in some form. Holliday intermediates have been observed in vivo in bacteria and in bacteriophageDNA (Fig.
24-28b). Note that there are two ways to cleave or "resolve"the Holliday intermediate so that the process is conservative, that is,so that the two products contain the same genes linked in the samelinear order as in the substrates. If cleaved one way, the DNA flankingthe heteroduplex region is recombined; if cleaved the other way, theflanking DNA is not recombined (Fig. 24-28a). Both outcomes are observed in vivo in both eukaryotes and prokaryotes.Homologous recombination as illustrated in Figure 24-28 is a veryelaborate process with subtle molecular consequences. To understandhow this process affects genetic diversity, it is important to note thathomologous does not necessarily mean identical. The two homologouschromosomes that are recombined may contain the same linear array843Chapter 24 DNA Metabolismof genes, but each chromosome may have slightly different base sequences in some of these genes.
In a human, for example, one chromosome may contain the normal gene for hemoglobin while the othercontains a hemoglobin gene with the sickle-cell mutation. The differences may represent no more than a change in a base pair or twoamong millions of identical base pairs. Although homologous recombination does not change the linear array of genes, it can determinewhich of the different versions (or alleles) of the genes are linked together on a single chromosome (Fig.
24-28).Recombination Requires Specific EnzymesEnzymes have been isolated from both prokaryotes and eukaryotesthat promote one or more steps of homologous recombination. Again,progress in both identifying and understanding these enzymes hasbeen greatest inE. coli. Important recombination enzymes are encodedby the recA, B, C, and D genes, and by the ruuC gene.
The recB, C, andD genes encode the RecBCD enzyme, which can initiate recombinationby unwinding DNA and occasionally cleaving one strand. The RecAprotein promotes all the central steps in the process: the pairing of twoDNAs, formation of Holliday intermediates, and branch migration asdescribed below. A novel class of nucleases that specifically cleave Holliday intermediates have also been isolated from bacteria and yeast.These nucleases are often called resolvases; the E. coli resolvase is theRuvC protein.The RecBCD enzyme binds to linear DNA at one end and uses theenergy of ATP to travel along the helix, unwinding the DNA ahead andrewinding it behind (Fig.
24-30). Rewinding is slower than unwindingso that a single-stranded bubble is gradually formed and enlarged. Thesingle strands in the bubble are cut when the enzyme encounters acertain sequence called chi, (5')GCTGGTGG(3'). There are about 1,000of these sequences in the E. coli genome, and they have the effect ofincreasing the frequency of recombination in the regions where theyoccur.
Sequences that enhance recombination frequency have alsobeen identified in several other organisms.The RecA protein is unusual among proteins involved in DNA metabolism in that its active form is an ordered, helical filament thatassembles cooperatively on DNA and can involve thousands of RecAmonomers (Fig.
24-31). Formation of this filament normally occurs onsingle-stranded DNA such as that produced by the RecBCD enzyme.The filament will also form on a duplex DNA with a single-strandedgap, in which case the first RecA monomers bind to the single-strandedDNA in the gap and then filament assembly rapidly envelops theneighboring duplex.RecBCDenzymechiATP- ADP + ^Helicase activity of enzyme producessingle-stranded bubbles.On reaching a chi sequence, nucleaseactivity cleaves the adjacent single strand.OH 3"Figure 24-30 Helicase and nuclease activities ofthe RecBCD enzyme. Unwinding of DNA ahead ofthe moving enzyme and slower rewinding behindcreate single-stranded bubbles. One strand iscleaved when the enzyme encounters a chi sequence.
Movement of the enzyme requires ATPhydrolysis. This enzyme is believed to help initiatehomologous genetic recombination in E. coli.Figure 24-31 (a) Nucleoprotein filament of RecAprotein on single-stranded DNA, as seen with theelectron microscope. The striations make evidentthe right-handed helical structure of the filament,(b) A computer enhancement of the structure seenwith the electron microscope.(\%\848Part IV Information PathwaysInversionSites of exchangeDeletion and insertion(reverse reactions)InsertionDeletionFigure 24-36 Possible outcome of site-specific recombination, depending on location and orientationof recombination sites (red and blue) in a doublestranded DNA molecule. Inversion and deletion andinsertion are illustrated. Orientation here refers tothe order of nucleotides in the recombination site,not the 5'—>3' direction.The sequences of recombination sites recognized by these recombinases are partially asymmetric (nonpalindromic), and the two recombining sites are aligned in the same orientation for reaction by therecombinase.
The reaction can have several outcomes, depending onthe relative location and orientation of the recombination sites (Fig.24-36). If the two sites are on the same DNA molecule the reaction willresult in either inversion or deletion of the DNA between them, depending on whether the sites have the opposite or the same orientation, respectively.
If the sites are on different DNAs the recombinationis intermolecular, and an insertion reaction occurs if one or both ofthese DNAs is circular. Some systems are highly specific for one ofthese reactions (e.g., inversions) and will not act on sites in the wrongrelative orientation.The first site-specific recombination system identified and studiedin vitro was that encoded by the bacteriophage A. When A phage DNAenters an E. coli cell, a complex series of regulatory events ensues thatcommits the DNA to one of two fates: either it is replicated and used toproduce more bacteriophages (in which case the host cell is destroyed),or it is integrated into the host chromosome where it can be replicatedpassively along with the host chromosome for many cell generations.Integration is accomplished by a phage-encoded recombinase calledthe A integrase, acting at recombination sites (attachment sites in thebacteriophage A system) on the phage and bacterial DNAs called attPand attB, respectively (Fig.
24-37). Several auxiliary proteins also areused in this reaction, some encoded by the bacteriophage and others bythe bacterial host cell. Note that a site-specific recombination reaction(Fig. 24-35) is chemically symmetric in terms of the chemical bondspresent before and after, and it should have an equilibrium constant of1.0.
A major function of the auxiliary proteins in A integration is toalter this equilibrium by permitting integration and/or preventing thereverse reaction (excision). The mechanism by which this is accomplished is not understood in detail.
When the bacteriophage DNA musteventually be excised from the chromosome (which occurs when thecell is subjected to a variety of environmental stresses), the sitespecific excision reaction uses a different set of auxiliary proteins (Fig.24-37).The use of site-specific recombination to regulate gene expressionwill be considered in Chapter 27.Immunoglobulin Genes Are Assembled by RecombinationAn important example of a programmed recombination event that occurs during development is the generation of immunoglobulin genesfrom gene segments that are separate in the genome. Immunoglobulins (or antibodies), produced by B lymphocytes, are the foot soldiers ofthe vertebrate immune system—the molecules that bind to infectiousagents and all substances foreign to the organism. A mammal such asa human is capable of producing many millions of different antibodieswith distinct binding specificities.
However, the human genome contains only about 100,000 genes. Recombination allows an organism toproduce an extraordinary diversity of antibodies from a relativelysmall amount of DNA-coding capacity.Vertebrates generally produce multiple classes of immunoglobulins. To illustrate how antibody diversity is generated, we will focus onthe immunoglobulin G (IgG) class from humans. Immunoglobulinsconsist of two heavy and two light polypeptide chains (Fig. 24-38a).Chapter 24 DNA MetabolismBacterial attachmentsite (attB)849atthPhageattachmentsite (attP)oA, PhageDNAIntegratedX phage DNA(prophage)Integration:X integrase (INT)IHFattRExcision:X integrase (INT)IHFFIS + XISE.
coli chromosomeFigure 24-37 The integration and excision of bacteriophage A DNA at the chromosomal target site.The attachment site on the A phage DNA (attP)shares only 15 base pairs of complete homologywith the bacterial site (attB) in the region of thecrossover. The reaction generates two new attachment sites (attR and attL) flanking the integratedphage DNA. The recombinase is the A integrase orINT protein.
Integration and excision use differentattachment sites and different auxiliary proteins.Excision uses the proteins XIS, encoded by the bacteriophage, and FIS, encoded by the bacterium.Both reactions require the protein IHF (integrationhost /actor), encoded by the bacterium.V segments(1 to-300)J segmentsC segmentGerm-lineDNARecombination results indeletion of DNA betweenV and J segments.Antigen-bindingdomainsMature lightchain geneS3TranscriptionVariableregionJoiningregionLightchainConstantregionsIPrimarytranscriptSequences betweenJ4 and C are removedby mRNA splicing.Translation11VariableteregionProtein folding and assemblyFigure 24-38 (a) Polypeptide chains in an immunoglobulin.
(b) Recombination of the V and J genesegments of the human IgG kappa light chain in aprocess designed to generate antibody diversity.The recombination reaction occurs in the first step.The C segment is joined to the J segment in a posttranscriptional splicing reaction (Chapter 25).DNA ofB lymphocyteLProcessedmRNALight chainpolypeptideConstantregionAntibodymolecule850Part IV Information PathwaysEach chain has a variable region with a sequence that differs greatlyfrom one immunoglobulin to the next, and another region that is virtually constant within a class of immunoglobulins. There are also twodistinct families of light chains, called kappa and lambda, which differsomewhat in the sequences of their constant regions. For each of thethree types of polypeptide chain (heavy chain, and kappa or lambdalight chain), diversity in the variable regions is generated by a similarmechanism.
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