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Mostrecombination events are conservative in the sense that genetic information is neither lost nor gained. Indeed, with a closer look at a recombination event, one often finds a DNA repair or gene regulation processin disguise.Special emphasis is given in this chapter to the enzymes that catalyze these processes. They are well worth getting acquainted with if forno other reason than their everyday use as reagents in a wide range ofmodern biochemical technologies. Because many of the seminal discoveries in DNA metabolism have been made with E.
coli, the well-understood enzymes obtained from this bacterium are generally used here toillustrate the ground rules. A quick look at the relevant genes on theE. coli genetic map (Fig. 24-1) provides just a hint of what is to come.Mismatch repair protein mutLSingle-strand DNA binding protein ssbDNA repair uvrAPrimosome component dnaBRNA polymerase frpoBisubunits \rpoCjFigure 24—1 A map of the E. coli chromosome,showing the relative positions of genes encodingsome of the proteins important in DNA metabolism.The number of known genes involved provides ahint of the complexity of these processes.
The numbers 0 to 100 denote a genetic measurement calledminutes, with each minute corresponding to about40,000 base pairs. The acronyms consisting of threelowercase letters generally reflect some aspect ofthe gene's function. These include mut, mutagenesis; dna, DNA replication; pol, DNA po/ymerase;rpo, #NA polymerase; uvr, UV-resistance; rec, recombination; ter, termination of replication; ori, origin of replication; dam, DNA adenine methylation;lig, DNA ligase; cou, cowmermycin resistance; andnal,rcaZidixicacid resistance (coumermycin andnalidixic acid inhibit DNA replication by binding tothe subunits of DNA gyrase encoded by thesegenes).dnaC Primosome componentdnaJ, dnaKpolB DNA polymerase IIDNA polymerase I polADNA hehcase/mismatch repair uvrDmutUdnaPmutTpolC (dnaE) DNA polymerase IIImutDHelicase 3'—>5' repDNA polymerase accessory proteini Replication origin )|oriCReplication initiation dnaADNA gyrase subunit couuvrB DNA repairinduced mutagenesisMethylation damRNA polymerase frpoAlsubunits \rpoDjPnmase dnaG| ter | ' Replication teimination>dualMismatch repair proteinsruuC Recombination and recombmational repairuvrC DNA repairRecombinationand recombmational -s recB >repair [recBJDNA ligase ligrecA Recombination andrecombinational repair815nalA DNA gyrase subunit816Part IV Information PathwaysBefore moving on to replication, we must entertain two short digressions.
The first concerns the use of acronyms in naming genes andproteins. Bacterial genetics is a powerful tool that has facilitated muchof the work described in this chapter. Bacterial genes that affect agiven cellular process such as replication often have been identifiedbefore the roles of their protein products were understood. By convention, acronyms used to identify bacterial (and sometimes eukaryotic)genes are generally three lowercase, italicized letters that reflect function, such as dna, uvr, or rec for genes that affect DNA replication,resistance to the damaging effects of UV radiation, or recombination,respectively.
In the case of multiple genes that affect the same process,the designation A, B, C, etc., is added, usually reflecting the temporalorder of gene discovery rather than a reaction sequence. In most cases,the protein product of each gene is ultimately isolated and characterized. Sometimes the product is identified as a previously isolated protein. The dnaE gene, for example, was found to encode the polymerizing subunit of DNA polymerase III; consequently, the dnaE gene wasrenamed polC to reflect that function more clearly. In many cases theprotein product has turned out to be novel, with an activity not easilydescribed by a simple enzyme name.
In a practice that can be confusing, these proteins often retain the name of their genes; for example,the products of the dnaA and rec A genes are simply called the DnaAand RecA proteins, respectively. Many examples of this practice arefound in this chapter. Here we use the convention that names in italicsrefer to genes or important DNA sequences, and roman type is usedwhen the name refers to a protein.*The second digression is needed to introduce enzymes that degradeDNA rather than synthesize it, because directed DNA degradationplays a significant role in all of the processes described in this chapter.These enzymes are called nucleases or, alternatively, DNases if theyare specific for DNA. Every cell contains several different nucleases,and these fall into two broad classes: exonucleases and endonucleases.Exonucleases degrade DNA from one end of the molecule.
Many arespecific for degradation in either the 5'-»3' or 3'->5' direction; that is,they remove nucleotides specifically from the 5' or 3' end, respectively,of one strand of a double-stranded nucleic acid (see Fig. 12-7). Endonucleases act in the interior of nucleic acids, reducing them to smallerand smaller fragments. A few exonucleases and endonucleases degrade only single-stranded DNA. There are also a few importantclasses of endonucleases that cleave only at specific nucleotide sequences (e.g., the restriction endonucleases considered in Chapter 28).Many types of nucleases will be encountered in this and subsequentchapters.DNA ReplicationLong before the structure of DNA became known, scientists had wondered first at the ability of organisms to create reasonable copies ofthemselves, and later at the ability of cells to produce many identical* For eukaryotic proteins, these naming conventions are somewhat different and varysufficiently from one organism to the next that no single convention can be presentedChapter 24 DNA Metabolismcopies of large and complex macromolecules.
Speculation about theseproblems centered around the concept of a template. The moleculartemplate had to be a surface upon which molecules could be lined up ina specific order and joined to create a macromolecule with a uniquestructure and function.The process of DNA replication provided the first biological example of the use of a molecular template to guide the synthesis of a macromolecule. The 1940s brought the revelation that DNA was the geneticmolecule, but not until James Watson and Francis Crick deduced itsstructure did it become clear how DNA could act as a template for thereplication and transmission of genetic information. One strand is thecomplement of the other.
The strict base-pairing rules mean that theuse of one strand as a template will result in another strand with apredictable, complementary sequence.The fundamental properties of the DNA replication process andthe mechanisms used by the enzymes that catalyze it have proven to beessentially identical in all organisms. This mechanistic unity will be amajor theme as we proceed from general properties of the replicationprocess to E. coli replication enzymes and finally to replication ineukaryotes.817DNA extracted and centrifugedto equilibrium in CsCldensity gradiento"Heavy"DNA( 15 N) -Original parentmolecule"Hybrid" DNADNA Replication Is Governed by a Set of Fundamental Rules(15 N /14 N ) -First-generationdaughter moleculesDNA Replication Is Semiconservative If each DNA strand serves asa template for the synthesis of a new strand, two new DNA moleculeswill result, each with one new strand and one old strand.
This is calledsemiconservative replication.The hypothesis of semiconservative replication was proposed byWatson and Crick soon after publication of their paper on the structureof DNA; the theory was proven in ingeniously designed experiments byMatthew Meselson and Franklin Stahl in 1957 (Fig. 24-2). Meselsonand Stahl grew E. coli cells for many generations in a medium in whichthe sole nitrogen source (NH4C1) contained 15N, the "heavy" isotope ofnitrogen, instead of the normal, more abundant "light" isotope 14N.
TheDNA isolated from these cells had a density about 1% greater than thatof normal [14N]DNA. Although this is only a small difference, a mixtureof heavy [15N]DNA and light [14N]DNA can be separated by centrifugation to equilibrium in a cesium chloride density gradient.The E. coli cells grown in the 15N medium were transferred to afresh medium containing only the 14N isotope, where they were allowed to grow until the cell population had just doubled. The DNAisolated from these first-generation cells formed a single band in theCsCl gradient at a position indicating that the double-helical DNAs ofthe daughter cells were hybrids containing one new 14N strand and oneparental 15N strand (Fig.
24-2).This result argued against conservative replication, an alternativehypothesis in which one progeny DNA molecule would consist of twonewly synthesized DNA strands and the other would contain the twoparental strands; this would never yield hybrid DNA molecules in theMeselson-Stahl experiment.
The semiconservative replication hypothesis was further supported in the next step of the experiment. Cellswere allowed to double in number again in the 14N medium, and theisolated DNA product of this second cycle of replication exhibited twobands, one having a density equal to that of light DNA and the otherhaving the density of the hybrid DNA observed after the first cell doubling."Light"DNA( 14 N)"Hybrid" DNASecond-generationdaughter moleculesFigure 24—2 The Meselson—Stahl experiment wasdesigned to distinguish between two alternativeDNA replication mechanisms.
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