The Molecular Basis of Inheritance

Transcription

1 Chapter 16 The Mlecular Basis f Inheritance Lecture Outline Overview: Life s Operating Instructins In April 1953, James Watsn and Francis Crick shk the scientific wrld with an elegant duble-helical mdel fr the structure f dexyribnucleic acid, r DNA. Yur genetic endwment is yur DNA, cntained in the 46 chrmsmes yu inherited frm yur parents and the mitchndria yu inherited frm yur mther. Nucleic acids are unique in their ability t direct their wn replicatin. The resemblance f ffspring t their parents depends n the precise replicatin f DNA and its transmissin frm ne generatin t the next. It is this DNA prgram that directs the develpment f yur bichemical, anatmical, physilgical, and (t sme extent) behaviral traits. Cncept 16.1 DNA is the genetic material. The search fr genetic material led t DNA. After T. H. Mrgan s grup shwed that genes are lcated n chrmsmes, the tw cnstituents f chrmsmes prteins and DNA were became the candidates fr the genetic material. Until the 1940s, the great hetergeneity and specificity f functin f prteins seemed t indicate that prteins were the genetic material. Hwever, this was nt cnsistent with experiments with micrrganisms, such as bacteria and viruses. The discvery f the genetic rle f DNA began with research by Frederick Griffith in Griffith studied Streptcccus pneumniae, a bacterium that causes pneumnia in mammals. One strain was harmless. The ther strain was pathgenic (disease-causing). Griffith mixed a heat-killed, pathgenic strain with a live, harmless strain f bacteria and injected this int a muse. The muse died, and Griffith recvered the pathgenic strain frm the muse s bld. Griffith called this phenmenn transfrmatin, a phenmenn nw defined as a change in gentype and phentype due t the assimilatin f external DNA by a cell. Fr the next 14 years, American bacterilgist Oswald Avery tried t identify the transfrming substance. Lecture Outline fr Campbell/Reece Bilgy, 8 th Editin, Pearsn Educatin, Inc. 16-1

2 Avery fcused n the three main candidates: DNA, RNA, and prtein. Avery brke pen the heat-killed, pathgenic bacteria and extracted the cellular cntents. He used specific treatments that inactivated each f the three types f mlecules. He then tested the ability f each sample t transfrm harmless bacteria. Only DNA was able t bring abut transfrmatin. Finally, in 1944, Oswald Avery, Maclyn McCarty, and Clin MacLed annunced that the transfrming substance was DNA. Still, many bilgists were skeptical because prteins were cnsidered better candidates fr the genetic material. There was als a belief that the genes f bacteria culd nt be similar in cmpsitin and functin t thse f mre cmplex rganisms. Further evidence that DNA was the genetic material came frm studies that tracked the infectin f bacteria by viruses. Viruses cnsist f DNA (r smetimes RNA) enclsed in a prtective cat f prtein. T replicate, a virus infects a hst cell and takes ver the cell s metablic machinery. Viruses that specifically attack bacteria are called bacteriphages r just phages. In 1952, Alfred Hershey and Martha Chase shwed that DNA was the genetic material f the phage T2. The T2 phage, cnsisting almst entirely f DNA and prtein, attacks Escherichia cli (E. cli), a cmmn intestinal bacteria f mammals. This phage can quickly turn an E. cli cell int a T2-prducing factry that releases phages when the cell ruptures. T determine the surce f genetic material in the phage, Hershey and Chase designed an experiment in which they culd label prtein and DNA and then track which entered the E. cli cell during infectin. They grew ne batch f T2 phage in the presence f radiactive sulfur, marking the prteins but nt DNA. They grew anther batch in the presence f radiactive phsphrus, marking the DNA but nt prteins. They allwed each batch t infect separate E. cli cultures. Shrtly after the nset f infectin, Hershey and Chase spun the cultured infected cells in a blender, shaking lse any parts f the phage that remained utside the bacteria. The mixtures were spun in a centrifuge, which separated the heavier bacterial cells in the pellet frm the lighter free phages and parts f phages in the liquid supernatant. They then tested the pellet and supernatant f the separate treatments fr the presence f radiactivity. Hershey and Chase fund that when the bacteria had been infected with T2 phages that cntained radilabeled prteins, mst f the radiactivity was in the supernatant that cntained phage particles, nt in the pellet with the bacteria. When they examined the bacterial cultures with T2 phage that had radilabeled DNA, mst f the radiactivity was in the pellet with the bacteria. Hershey and Chase cncluded that the injected DNA f the phage prvides the genetic infrmatin that makes the infected cells prduce new viral DNA and prteins t assemble int new viruses. Lecture Outline fr Campbell/Reece Bilgy, 8 th Editin, Pearsn Educatin, Inc. 16-2

3 The fact that a cell dubles its amunt f DNA prir t mitsis and then distributes the DNA equally t each daughter cell prvided sme circumstantial evidence that DNA was the genetic material in eukarytes. Similar circumstantial evidence came frm the bservatin that diplid sets f chrmsmes have twice as much DNA as the haplid sets in gametes f the same rganism. By 1947, Erwin Chargaff had develped a series f rules based n a survey f DNA cmpsitin in rganisms. He already knew that DNA was a plymer f nucletides cnsisting f a nitrgenus base, dexyribse, and a phsphate grup. The bases culd be adenine (A), thymine (T), guanine (G), r cytsine (C). Chargaff nted that the DNA cmpsitin varies frm species t species. In any ne species, the fur bases are fund in characteristic, but nt necessarily equal, ratis. He als fund a peculiar regularity in the ratis f nucletide bases, knwn as Chargaff s rules. In all rganisms, the number f adenines is apprximately equal t the number f thymines (%T = %A). The number f guanines is apprximately equal t the number f cytsines (%G = %C). Human DNA is 30.3% adenine, 30.3% thymine, 19.5% guanine, and 19.9% cytsine. The basis fr these rules remained unexplained until the discvery f the duble helix. Watsn and Crick discvered the duble helix by building mdels t cnfrm t X-ray data. By the beginning f the 1950s, the race was n t mve frm the structure f a single DNA strand t the three-dimensinal structure f DNA. Amng the scientists wrking n the prblem were Linus Pauling in Califrnia and Maurice Wilkins and Rsalind Franklin in Lndn. Wilkins and Franklin used X-ray crystallgraphy t study the structure f DNA. In this technique, X-rays are diffracted as they pass thrugh aligned fibers f purified DNA. The diffractin pattern can be used t deduce the three-dimensinal shape f mlecules. James Watsn learned frm this research that DNA is helical, and he deduced the width f the helix and the spacing f nitrgenus bases. The width f the helix suggested that it is made up f tw strands, cntrary t a threestranded mdel that Linus Pauling had recently prpsed. Watsn and his clleague Francis Crick began t wrk n a mdel f DNA with tw strands, the duble helix. Frm an unpublished annual reprt summarizing Franklin s wrk, Watsn and Crick knew Franklin had cncluded that sugar-phsphate backbnes were n the utside f the duble helix. Using mlecular mdels made f wire, they placed the sugar-phsphate chains n the utside and the nitrgenus bases n the inside f the duble helix. This arrangement put the relatively hydrphbic nitrgenus bases in the mlecule s interir, away frm the surrunding aqueus slutin. In their mdel, the tw sugar-phsphate backbnes are antiparallel, with the subunits running in ppsite directins. Lecture Outline fr Campbell/Reece Bilgy, 8 th Editin, Pearsn Educatin, Inc. 16-3

4 The sugar-phsphate chains f each strand are like the side rpes f a rpe ladder. Pairs f nitrgenus bases, ne frm each strand, frm rungs. The ladder frms a twist every ten bases. The nitrgenus bases are paired in specific cmbinatins: adenine with thymine and guanine with cytsine. Pairing like nucletides did nt fit the unifrm diameter indicated by the X-ray data. A purine-purine pair is t wide, and a pyrimidine-pyrimidine pair is t shrt. Only a pyrimidine-purine pair prduces the 2-nm diameter indicated by the X-ray data. In additin, Watsn and Crick determined that chemical side grups f the nitrgenus bases frm hydrgen bnds, cnnecting the tw strands. Based n details f their structure, adenine frms tw hydrgen bnds nly with thymine, and guanine frms three hydrgen bnds nly with cytsine. This finding explained Chargaff s rules. The base-pairing rules dictate the cmbinatins f nitrgenus bases that frm the rungs f DNA. The rules d nt restrict the sequence f nucletides alng each DNA strand. The linear sequence f the fur bases can be varied in cuntless ways. Each gene has a unique rder f nitrgenus bases. In April 1953, Watsn and Crick published a succinct, ne-page paper in Nature reprting their duble helix mdel f DNA. Cncept 16.2 Many prteins wrk tgether in DNA replicatin and repair. The specific pairing f nitrgenus bases in DNA was the flash f inspiratin that led Watsn and Crick t the crrect duble helix. The pssible mechanism fr the next step, the accurate replicatin f DNA, was clear t Watsn and Crick frm their duble helix mdel. During DNA replicatin, base pairing enables existing DNA strands t serve as templates fr new cmplementary strands. In a secnd paper, Watsn and Crick published their hypthesis fr hw DNA replicates. Because each strand is cmplementary t the ther, each can frm a template when separated. The rder f bases n ne strand can be used t add cmplementary bases and therefre duplicate the pairs f bases exactly. When a cell cpies a DNA mlecule, each strand serves as a template fr rdering nucletides int a new cmplementary strand. One at a time, nucletides line up alng the template strand accrding t the base-pairing rules. The nucletides are linked t frm new strands. Watsn and Crick s mdel semicnservative replicatin predicts that when a duble helix replicates, each f the daughter mlecules has ne ld strand and ne newly made strand. Tw cmpeting mdels the cnservative mdel and the dispersive mdel were als prpsed. Lecture Outline fr Campbell/Reece Bilgy, 8 th Editin, Pearsn Educatin, Inc. 16-4

5 Experiments in the late 1950s by Matthew Meselsn and Franklin Stahl supprted the semicnservative mdel prpsed by Watsn and Crick ver the ther tw mdels. Meselsn and Stahl labeled the nucletides f the ld strands with a heavy istpe f nitrgen ( 15 N) and the new nucletides with a lighter istpe ( 14 N). Replicated strands culd be separated by density in a centrifuge. Each mdel the semicnservative mdel, the cnservative mdel, and the dispersive mdel made a specific predictin abut the density f replicated DNA strands. The first replicatin in the 14 N medium prduced a band f hybrid ( 15 N- 14 N) DNA, eliminating the cnservative mdel. A secnd replicatin prduced bth light and hybrid DNA, eliminating the dispersive mdel and supprting the semicnservative mdel. A large team f enzymes and ther prteins carries ut DNA replicatin. It takes E. cli less than an hur t cpy each f the 4.6 millin base pairs in its single chrmsme and divide t frm tw identical daughter cells. A human cell can cpy its 6 billin base pairs and divide int daughter cells in nly a few hurs. This prcess is remarkably accurate, with nly ne errr per 10 billin nucletides. Mre than a dzen enzymes and ther prteins participate in DNA replicatin. Much mre is knwn abut replicatin in bacteria than in eukarytes. The prcess appears t be fundamentally similar fr prkarytes and eukarytes. The replicatin f a DNA mlecule begins at special sites called rigins f replicatin. In bacteria, this site is a specific sequence f nucletides that is recgnized by the replicatin enzymes. These enzymes separate the strands, frming a replicatin bubble. Replicatin prceeds in bth directins until the entire mlecule is cpied. In eukarytes, there may be hundreds r thusands f rigin sites per chrmsme. At the rigin sites, the DNA strands separate, frming a replicatin bubble with replicatin frks at each end, where the parental strands f DNA are being unwund. The replicatin bubbles elngate as the DNA is replicated, and eventually fuse. Several kinds f prteins participate in the unwinding f parental strands f DNA. Helicase untwists the duble helix and separates the template DNA strands at the replicatin frk. This untwisting causes tighter twisting ahead f the replicatin frk, and tpismerase helps relieve this strain. Single-strand binding prteins keep the unpaired template strands apart during replicatin. The parental DNA strands are nw available t serve as templates fr the synthesis f new cmplementary DNA strands. The enzymes that synthesize cannt initiate synthesis f a plynucletide. These enzymes can nly add nucletides t the end f an existing chain that is basepaired with the template strand. Lecture Outline fr Campbell/Reece Bilgy, 8 th Editin, Pearsn Educatin, Inc. 16-5

6 The initial nucletide chain is a shrt stretch f RNA called a primer, synthesized by the enzyme primase. Primase can start an RNA chain frm a single RNA nucletide, adding RNA nucletides ne at a time, using the parental DNA strand as a template. The cmpleted primer is generally five t ten nucletides lng. The new DNA strand starts frm the 3 end f the RNA primer. As nucletides align with cmplementary bases alng the template strand, they are added t the grwing end f the new strand by the plymerase. Enzymes called DNA plymerases catalyze the synthesis f new DNA by adding nucletides t a preexisting chain. In E. cli, tw different DNA plymerases play majr rles in replicatin: DNA plymerase III and DNA plymerase I. In eukarytes, at least 11 different DNA plymerases have been identified s far. Mst DNA plymerases require a primer and a DNA template strand, alng which cmplementary DNA nucletides line up. The rate f elngatin is abut 500 nucletides per secnd in bacteria and 50 per secnd in human cells. Each nucletide that is added t a grwing DNA strand cmes frm a nucleside triphsphate, a nucleside (a nitrgenus base and a dexyribse sugar) and a triphsphate tail. ATP is a nucleside triphsphate with ribse instead f dexyribse. Like ATP, the triphsphate mnmers used fr DNA synthesis are chemically reactive, partly because their triphsphate tails have an unstable cluster f negative charge. As each nucletide is added t the grwing end f a DNA strand, the last tw phsphate grups are hydrlyzed t frm pyrphsphate. The exergnic hydrlysis f pyrphsphate t tw inrganic phsphate mlecules drives the plymerizatin f the nucletide t the new strand. The strands in the duble helix are antiparallel. The sugar-phsphate backbnes run in ppsite directins. Each DNA strand has a 3 end with a free hydrxyl grup attached t dexyribse and a 5 end with a free phsphate grup attached t dexyribse. The 5 3 directin f ne strand runs ppsite the 3 5 directin f the ther strand. DNA plymerases can add nucletides nly t the free 3 end f a grwing DNA strand. A new DNA strand can elngate nly in the 5 3 directin. Let s cnsider a replicatin frk. Alng ne template strand, DNA plymerase III can synthesize a cmplementary strand cntinuusly by elngating the new DNA in the mandatry 5 3 directin. DNA plymerase III sits in the replicatin frk n that template strand and cntinuusly adds nucletides t the new cmplementary strand as the frk prgresses. The DNA strand made by this mechanism, called the leading strand, requires a single primer. The ther parental strand (5 3 int the frk), the lagging strand, is cpied away frm the frk. Lecture Outline fr Campbell/Reece Bilgy, 8 th Editin, Pearsn Educatin, Inc. 16-6

7 Unlike the leading strand, which elngates cntinuusly, the lagging stand is synthesized as a series f shrt segments called Okazaki fragments. Okazaki fragments are abut 1,000 t 2,000 nucletides lng in E. cli and 100 t 200 nucletides lng in eukarytes. Anther enzyme, DNA ligase, eventually jins the sugar-phsphate backbnes f the Okazaki fragments t frm a single DNA strand. Althugh nly ne primer is required n the leading strand, each Okazaki fragment n the lagging strand must be primed separately. Anther DNA plymerase, DNA plymerase I, replaces the RNA nucletides f the primers with DNA versins, adding them ne by ne t the 3 end f the adjacent Okazaki fragment. DNA plymerase I cannt jin the final nucletide f the replacement DNA segment t the first DNA nucletide f the Okazaki fragment whse primer was just replaced. DNA ligase jins the sugar-phsphate backbnes f all the Okazaki fragments int a cntinuus DNA strand. T summarize: At the replicatin frk, the leading strand is cpied cntinuusly int the frk frm a single primer. The lagging strand is cpied away frm the frk in shrt segments, each requiring a new primer. It is cnventinal and cnvenient t think f the DNA plymerase mlecules as mving alng a statinary DNA template. In reality, the varius prteins invlved in DNA replicatin frm a single large cmplex, a DNA replicatin machine. Many prtein-prtein interactins facilitate the efficiency f this machine. Fr example, by interacting with ther prteins at the frk, primase slws the prgress f the replicatin frk and crdinates the rate f replicatin n the leading and lagging strands. The DNA replicatin machine is prbably statinary during the replicatin prcess. In eukarytic cells, multiple cpies f the machine may anchr t the nuclear matrix, a framewrk f fibers extending thrugh the interir f the nucleus. Tw DNA plymerase mlecules, ne n each template strand, reel in the parental DNA and extrude newly made daughter DNA mlecules. The lagging strand is lped back thrugh the cmplex. When a DNA plymerase cmpletes synthesis f an Okazaki fragment and dissciates, it desn t have far t travel t reach the primer fr the next fragment, near the replicatin frk. This lping f the lagging strand enables mre Okazaki fragments t be synthesized in less time. Enzymes prfread DNA during its replicatin and repair damage in existing DNA. Mistakes during the initial pairing f template nucletides and cmplementary nucletides ccur at a rate f ne errr per 100,000 base pairs. DNA plymerase prfreads each new nucletide against the template nucletide as sn as it is added. If there is an incrrect pairing, the enzyme remves the wrng nucletide and then resumes synthesis. Lecture Outline fr Campbell/Reece Bilgy, 8 th Editin, Pearsn Educatin, Inc. 16-7

8 The final errr rate is nly ne mistake per 10 billin nucletides. Mismatched nucletides that are missed by DNA plymerase r mutatins that ccur after DNA synthesis is cmpleted can ften be repaired. In mismatch repair, special enzymes fix incrrectly paired nucletides. A hereditary defect in ne f these enzymes is assciated with a frm f cln cancer. Incrrectly paired r altered nucletides can als arise after replicatin. DNA mlecules are cnstantly subjected t ptentially harmful chemical and physical agents. Reactive chemicals, radiactive emissins, X-rays, ultravilet light, and mlecules in cigarette smke can change nucletides in ways that can affect encded genetic infrmatin. DNA bases may underg spntaneus chemical changes under nrmal cellular cnditins. Each cell cntinuusly mnitrs and repairs its genetic material, with 100 repair enzymes knwn in E. cli and mre than 130 repair enzymes identified in humans. Many cellular systems fr repairing incrrectly paired nucletides use a mechanism that relies n the base-paired structure f DNA. A nuclease cuts ut a segment f a damaged strand, and the resulting gap is filled in with nucletides, using the undamaged strand as a template. One such DNA repair system is called nucletide excisin repair. DNA repair enzymes in skin cells repair genetic damage caused by ultravilet light. Ultravilet light can prduce thymine dimers between adjacent thymine nucletides. This buckles the DNA duble helix and interferes with DNA replicatin. The imprtance f the prper functining f the enzymes that repair damage t DNA is clear frm the inherited disrder xerderma pigmentsum. Individuals with this disrder are hypersensitive t sunlight. Mutatins in their skin cells are left uncrrected and cause skin cancer. The ends f DNA mlecules are replicated by a special mechanism. Limitatins f DNA plymerase create prblems fr the linear DNA f eukarytic chrmsmes. The usual replicatin machinery prvides n way t cmplete the 5 ends f daughter DNA strands. Repeated runds f replicatin prduce shrter and shrter DNA mlecules. Prkarytes d nt have this prblem because they have circular DNA mlecules withut ends. The ends f eukarytic chrmsmal DNA mlecules, the telmeres, have special nucletide sequences. Telmeres d nt cntain genes. Instead, the DNA typically cnsists f multiple repetitins f ne shrt nucletide sequence. In human telmeres, this sequence is typically TTAGGG, repeated between 100 and 1,000 times. Telmeric DNA binds t prtein cmplexes t prtect genes frm being erded thrugh multiple runds f DNA replicatin. Lecture Outline fr Campbell/Reece Bilgy, 8 th Editin, Pearsn Educatin, Inc. 16-8

9 Telmeric DNA and specific prteins assciated with it als prevent the staggered ends f the daughter mlecule frm activating the cell s system fr mnitring DNA damage. Telmeres becme shrter during every rund f replicatin. Telmeric DNA tends t be shrter in the dividing smatic cells f lder individuals and in cultured cells that have divided many times. It is pssible that the shrtening f telmeres is smehw cnnected with the aging prcess f certain tissues and perhaps t aging in general. Eukarytic cells have evlved a mechanism t restre shrtened telmeres in germ cells, which give rise t gametes. If the chrmsmes f germ cells became shrter with every cell cycle, essential genes wuld eventually be lst. An enzyme called telmerase catalyzes the lengthening f telmeres in eukarytic germ cells, restring their riginal length. Telmerase is nt present in mst cells f multicellular rganisms. Therefre, the DNA f dividing smatic cells and cultured cells tends t becme shrter. Telmere length may be a limiting factr in the life span f certain tissues and f the rganism. Nrmal shrtening f telmeres may prtect rganisms frm cancer by limiting the number f divisins that smatic cells can underg. Cells frm large tumrs ften have unusually shrt telmeres because they have gne thrugh many cell divisins. Active telmerase has been fund in sme cancerus smatic cells. Active telmerase vercmes the prgressive shrtening that wuld eventually lead t self-destructin f the cancer. Immrtal strains f cultured cells are capable f unlimited cell divisin. Telmerase may prvide a useful target fr cancer diagnsis and chemtherapy. Cncept 16.3 A chrmsme cnsists f a DNA mlecule packed tgether with prteins. Mst bacteria have a single, circular, duble-stranded DNA mlecule, assciated with a small amunt f prtein. The bacterial chrmsme is very different frm a eukaryte chrmsme, which is a linear DNA mlecule assciated with a large amunt f prtein The chrmsmal DNA f E. cli cnsists f abut 4.6 millin nucletide pairs, representing abut 4,400 genes. Stretched ut, this DNA wuld be abut a millimeter lng, 500 times lnger than an E. cli cell. A bacterium has a dense regin f DNA called the nucleid. Within the nucleid, prteins cause the chrmsme t tightly cil and supercil, densely packing it s that it fills nly part f the cell. Each eukarytic chrmsme cntains a single linear DNA duble helix that, in humans, has an average f abut nucletide pairs. If stretched ut, such a DNA mlecule wuld be abut 4 cm lng, thusands f times the diameter f a cell nucleus. In the cell, eukarytic DNA is packaged with prtein t frm chrmatin. Lecture Outline fr Campbell/Reece Bilgy, 8 th Editin, Pearsn Educatin, Inc. 16-9

10 The packing f chrmatin varies during the curse f the cell cycle. It is highly extended during interphase. As a cell enters mitsis, the chrmatin cils and cndenses, frming shrt, thick metaphase chrmsmes. Althugh interphase chrmatin is generally much less cndensed than the chrmatin f mittic chrmsmes, it shws several f the same levels f higher-rder packing. The lped dmains f an interphase chrmsme are attached t the nuclear lamina, n the inside f the nuclear envelpe, and perhaps als t fibers f the nuclear matrix. These attachments may help rganize regins where the infrmatin in specific genes is actively cnverted t RNA and prtein mlecules that carry ut necessary cell functins. The chrmatin f each chrmsme ccupies a specific restricted area within the interphase nucleus, preventing tangling f the chrmatin fibers f different chrmsmes. Even during interphase, the centrmeres and telmeres f chrmsmes exist in a highly cndensed state similar t that seen in a metaphase chrmsme. This type f interphase chrmatin is called heterchrmatin, t distinguish it frm mre dispersed euchrmatin. Because f its cmpactin, heterchrmatin DNA is largely inaccessible t the machinery in the cell respnsible fr expressing the genetic infrmatin cded in the DNA. Mre lsely packed euchrmatin makes its DNA accessible t this machinery, s the genes present in euchrmatin can be expressed. The chrmsme is a dynamic structure that is cndensed, lsened, mdified, and remdeled as necessary fr varius cell prcesses, including mitsis, meisis, and gene expressin. Histnes are nt simply inert spls arund which the DNA is wrapped. Histnes underg chemical mdificatins that result in changes in chrmatin rganizatin. Lecture Outline fr Campbell/Reece Bilgy, 8 th Editin, Pearsn Educatin, Inc