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For example, the mutation rate of a gene specifically requiredfor cells to use the sugar lactose as an energy source can be determined whenthe cells are grown in the presenceof a different sugar,such as glucose.The fraction of damaged genes underestimates the actual mutation rate because manymutations are silent (for example, those that change a codon but not the aminoacid it specifies,or those that change an amino acid without affecting the activity of the protein coded for by the gene).
After correcting for these silent mutations, one finds that a single gene that encodes an average-sizedprotein (-103coding nucleotide pairs) accumulates a mutation (not necessarily one thatwould inactivate the protein) about once in about 106bacterial cell generations.stated differently, bacteria display a mutation rate of about 1 nucleotide changeper 10snucleotides per cell generation.Recently it has become possible to measure the germ line mutation ratedirectly in more complex, sexually reproducing organisms such as the nemotode C.
elegans.These worms, whose generation time is 4 days, were grown formany generations using their self-fertilization mode of reproduction (discussedin chapter 22).The DNA sequence of a large region of the genome was thendetermined for many different descendent worms and compared with that ofthe progenitor worm. This analysis showed, on average, tvvo new mutations(mostly short insertions and deletions) arise in the haploid genome each generation. lVhen the number of cell divisions needed to produce sperm and eggs istaken into account, the mutation rate is roughly I mutation per l0e nucleotidesper cell division, a rate remarkably similar to that in the asexuallyreproducing Ecoli described above.Direct measurement of the germ-line mutation rate in mammals is moredifficult, but indirect estimates can be obtained.
one way is to compare theamino acid sequences of the same protein in several species.The fraction ofthe amino acids that differ between any two species can then be comparedwith the estimated number of years since that pair of species diverged from acommon ancestor, as determined from the fossil record. using this method,one can calculate the number ofyears that elapse,on average,before an inherited change in the amino acid sequence of a protein becomes fixed in anorganism.
Because each such change usually reflects the alteration of a singlenucleotide in the DNA sequence of the gene encoding that protein, we can usethis value to estimate the average number of years required to produce a single, stable mutation in the gene.These calculations will nearly always substantially underestimate the actualthe proteinFbrinogen is activated to form ftbrin during blood clotting. Since thefunction of fibrinopeptides apparently does not depend on their amino acidsequence,fibrinopeptides can tolerate almost any amino acid change. Sequencecomparisons of the fibrinopeptides can therefore be used to estimate the mutation rate in the germ line.
As determined from these studies, a typical protein of400 amino acids will suffer an amino acid alteration roughly once every 200,000years.Another way to estimate mutation rates in humans is to use DNA sequencing to compare corresponding nucleotide sequences directry from Closelyrelated species in regions of the genome that do not appear to carry criticalinformation. As expected,such comparisons produce estimates of the mutationrate that agreewith those obtained from the fibrinopeptide studies.E. coli,worms and humans differ greatly in their modes of reproduction andin their generation times.
Yet,when the mutation rates of each are normalized toa single round of DNA replication, they are found to be similar: roughly Inucleotide change per lOe nucleotides each time that DNA is replicated.265THEMAINTENANCEOF DNASEQUENCESg e r m - l i n ec e l l sg e r m - l r n ec e l l sO*O*O*O*-zygoteO*O*O*O*-\,0.r/t\r / \/\oo3otttltlttcc oc ot 0csr'/\-o/\ot 0u uo 3cs o m a t i cc e l l sMOTHERDAUGHTERfor LifeasWe KnowltLowMutationRatesAre NecessarySince many mutations are deleterious, no species can afford to allow them toaccumulate at a high rate in its germ cells.Although the observed mutation frequency is low, it is neverthelessthought to limit the number of essentialproteinsthat any organism can encode to perhaps 50,000.By an extension of the sameargument, a mutation frequency tenfold higher would limit an organism toabout 5000 essential genes.
In this case, evolution would have been limited toorganisms considerably less complex than a fruit fly.The cells of a sexuallyreproducing organism are of two t)?es: germ cells andsomatic cells. The germ cells transmit genetic information from parent to offspring; the somatic cells form the body of the organism (Figure 5-l). We haveseen that germ cells must be protected against high rates of mutation to maintain the species.However,the somatic cells of multicellular organisms must alsobe protected from genetic change to safeguard each individual. Nucleotidechanges in somatic cells can give rise to variant cells, some of which, throughnatural selection, proliferate rapidly at the expense of the rest of the organism.In an extreme case,the result is an uncontrolled cell proliferation known as cancer, a diseasethat causesmore than 20% of the deaths each year in Europe andNorth America.
These deaths are due largely to an accumulation of changes inthe DNA sequences of somatic cells (discussed in Chapter 23). A significantincrease in the mutation frequency would presumably cause a disastrousincrease in the incidence of cancer by acceleratingthe rate at which somatic cellvariants arise.Thus, both for the perpetuation of a specieswith a large numberof genes (germ cell stability) and for the prevention of cancer resulting frommutations in somatic cells (somatic cell stability), multicellular organisms likeourselves depend on the remarkably high fidelity with which their DNAsequencesare replicated and maintained.SummaryIn all cells, DNA sequencesare meintained and replicated with hiSh ftdelity.
Themutation rate,approximatelyI nucleotidechangeper ld nucleotideseach time theD,n/Ais replicated, is roughty the same for organisms as different as bacteria andhumans. Becauseof this remarkableaccuracy,the sequenceof the human genome(approximatety3x 1d nucleotidepairs) is changedbyonly about 3 nucleotideseachtime a cell diuides.This allows most humans to pqssaccurategeneticinstructionsfrom one generation to the next, and also to auoid the changesin somatic cells thatlead to cancer.Figure5-1 Germ-linecellsand somaticcellscarry out fundamentally differentfunctions.In sexuallyreproducingorganisms,the germ-linecells(red)propagategeneticinformationinto thenext generation.Somaticcells(blue),arewhichform the body of the organism,for the survivalof germ-linenecessaryleaveanycellsbut do not themselvesprogeny.266Chapter5: DNAReplication,Repair,and RecombinationDNAREPLICATIONMECHANISMSAll organisms must duplicate their DNA with extraordinary accuracy beforeeach cell division.
In this section, we explore how an elaborate "replicationmachine" achievesthis accuracy,while duplicating DNA at rates as high as 1000nucleotides per second.Base-PairingUnderliesDNAReplicationand DNARepairAs introduced in chapter r, DNA templatingis the mechanism the cell uses tocopy the nucleotide sequence of one DNA strand into a complementary DNAsequence (Figure 5-2). This process entails the recognition of each nucleotide inthe DNA template strandby a free (unpolimerized) complementary nucleotide,and it requires the separation of the two strands of the DNA helix.
This separation exposesthe hydrogen-bond donor and acceptor groups on each DNA basefor base-pairing with the appropriate incoming free nucleotide, aligning it for itsenzlrne-catalyzed polyrnerization into a new DNA chain.The first nucleotide-polymerizing enzyme, DNA polymerase, was discovered in 1957.The free nucleotides that serve as substratesfor this enzyme werefound to be deoxyribonucleoside triphosphates, and their poll'rnerization intoDNA required a single-stranded DNA template. Figure 5-3 and Figure 5-4 illustrate the stepwise mechanism of this reaction.TheDNAReplicationForkls AsymmetricalDuring DNA replication inside a cell, each of the two original DNA strandsservesas a template for the formation of an entire new strand.
Becauseeach ofthe two daughters of a dividing cell inherits a new DNA double helix containingone original and one new strand (Figure S-5), the DNA double helix is said to bereplicated "semiconservatively" by DNA polymerase. How is this feat accomplished?daughter strands.t e m p l a t eS s t r a n dtrW5 strandr;4.."',*Li4t- *new 5' strandnew S strand5'strandp a r e n tD N A d o u b l eh e l i xWWt e m p l a t e5 ' s t r a n dFigure5-2 The DNAdouble helixactsas a templatefor its ownduplication.Becausethe nucleotideA will pairsuccessfullyonlywith Iand G only with C,eachstrandof DNAcan serveas a templateto specifythe sequenceof nucleotidesin its complementarystrandby DNAbasepairing.In this way,a double-helicalDNAmoleculecan be copiedprecisely.TEMPLATESTRANDooo- o - Pi l -i lot l- P ttlo-P-O-co-o-pyrophosphateincoming deoryribonucleosidetriphosphateO= p-O-offiMFigure5-3 The chemistryof DNA synthesis.The additionof adeoxyribonucleotideto the 3' end of a polynucleotidechain (the primerAsstrand)is the fundamentalreactionby which DNA is synthesized.shown,base-pairingbetweenan incomingdeoxyribonucleosidetriphosphateand an existingstrandof DNA (the templatestrand)guidesthe formationof the new strandof DNA and causesit to haveacomplementarynucleotidesequence.Initially, the simplest mechanism of DNA replication seemed to be the continuous growth of both new strands, nucleotide by nucleotide, at the replicationfork asit movesfrom one end of a DNA moleculeto the other.But becauseof theantiparallelorientation of the two DNA strandsin the DNA double helix (seeFigure 5-2), this mechanismwould require one daughter strand to polymerize inthe 5'-to-3' directionand the other in the 3'-to-5' direction.Sucha replicationfork would require two distinct types of DNA polymeraseen4/mes.However,allof the many DNA polymerasesthat havebeen discoveredcan slmthesizeonly inthe 5'-to-3'direction.HoW then, can a DNA strand grow in the 3'-to-5' direction?The answerwasfirst suggestedby the results of an experiment performed in the Iate 1960s.Researchersadded highly radioactiveaU-thymidine to dividing bacteria for afew seconds,so that only the most recently replicated DNA-that just behindthe replication fork-became radiolabeled.This experiment revealedthe transient existenceof pieces of DNA that were 1000-2000nucleotides long, nowChapter5: DNAReplication,Repair,and Recombination5'triphosphate3', r---rHO\IlypnmerstrandIncomtngdeoxyribonucleosidetriphosphate5'-to-3'directionofchaingrowthtemplatestrand(A)Incomrngdeoxynucleosidetriphosphatetemplatestrand5-POstTtONtNGOFINCOMINGprimer DEOXYNUCLEOSIDEStrand TRIPHOSPHATEq-,INCORPORATIONFOLLOWEDBY DNATRANSLOCATIONFigure5-4 DNA synthesiscatalyzedby DNA polymerase.(A)As indicated,DNA polymerasecatalyzesthestepwiseaddition of a deoxyribonucleotideto the 3rOH end of a polynucleotidechain,the primer strandthat is pairedto a secondtemplotestrand.The newly synthesizedDNAstrandthereforepolymerizesin the51to-3'directionas shownin the previousfigure.Becauseeachincomingdeoxyribonucleosidetriphosphatemust pair with the templatestrandto be recognizedby the DNA polymerase,this stranddetermineswhich of the four possibledeoxyribonucleotides(A,C,G,or T) will be added.The reactionisdriven by a large,favorablefree-energychange,causedby the releaseof pyrophosphateand itssubsequenthydrolysis(B)Theshapeof a DNApolymeraseto two moleculesof inorganicphosphate.molecule,as determinedby x-raycrystallography.RoughlyspeakingDNApolymerasesresemblea righthandin whichthe palm,fingers,and thumb graspthe DNAand form the activesite.In the sequenceshown,the correctpositioningof an incomingdeoxynucleosidetriphosphatecausesthe fingersof thepolymeraseto tighten,therebyinitiatingthe nucleotideaddition reaction.Dissociationof pyrophosphatecausesreleaseof the fingersand translocationof the DNAby one nucleotideso the activesiteof thepolymeraseis readyto receivethe next deoxynucleosidetriphosphate.InThe High Fidelityof DNAReplicationRequiresSeveratProofreadingMechanismsAs discussedabove,the fidelity of copying DNA during replication is such thatonly about I mistake occurs for every 10enucleotides copied.
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