The Molecular Basis of Inheritance

Transcription

1 MPBELL BIOLOY IN FOUS URRY IN WSSERMN MINORSKY REEE 13 he Molecular Basis of Inheritance Lecture Presentations by Kathleen Fitzpatrick and Nicole unbridge, Simon Fraser University SEOND EDIION Overview: Life s Operating Instructions In 1953, James Watson and Francis rick introduced an elegant double-helical model for the structure of deoxyribonucleic acid, or DN DN, the substance of inheritance, is the most celebrated molecule of our time Hereditary information is encoded in DN and reproduced in all cells of the body (DN replication) Figure

2 Figure 13.2 oncept 13.1: DN is the genetic material Early in the 20th century, the identification of the molecules of inheritance loomed as a major challenge to biologists he Search for the enetic Material: Scientific Inquiry When. H. Morgan s group showed that genes are located on chromosomes, the two components of chromosomes DN and protein became candidates for the genetic material he key factor in determining the genetic material was choosing appropriate experimental organisms he role of DN in heredity was worked out by studying bacteria and the viruses that infect them 2

3 Evidence hat DN an ransform Bacteria he discovery of the genetic role of DN began with research by Frederick riffith in 1928 riffith worked with two strains of a bacterium, one pathogenic and one harmless When he mixed heat-killed remains of the pathogenic strain with living cells of the harmless strain, some living cells became pathogenic He called this phenomenon transformation, now defined as a change in genotype and phenotype due to assimilation of foreign DN Figure 13.3 Experiment Living S cells (control) Living R cells (control) Heat-killed S cells (control) Mixture of heatkilled S cells and living R cells Results Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells 3

4 Later work by Oswald very and others identified the transforming substance as DN Many biologists remained skeptical, mainly because little was known about DN and they thought proteins were better candidates for the genetic material Evidence hat Viral DN an Program ells More evidence for DN as the genetic material came from studies of viruses that infect bacteria Such viruses, called bacteriophages (or phages), are widely used in molecular genetics research virus is DN (or RN) enclosed by a protective protein coat Viruses must infect cells and take over the cells metabolic machinery in order to reproduce nimation: Phage 2 Reproduction 4

5 Figure 13.4 Phage head DN ail sheath ail fiber enetic material Bacterial cell 100 nm In 1952, lfred Hershey and Martha hase showed that DN is the genetic material of a phage known as 2 o determine this, they designed an experiment showing that only the DN of the 2 phage, and not the protein, enters an E. coli cell during infection hey concluded that the injected DN of the phage provides the genetic information nimation: Hershey-hase Experiment 5

6 Figure 13.5 Experiment Batch 1: Radioactive sulfur ( 35 S) in phage protein Labeled phages infect cells. Radioactive protein gitation frees outside phage parts from cells. entrifuged cells form a pellet. Free phages and phage parts remain in liquid. Radioactivity (phage protein) found in liquid DN entrifuge Batch 2: Radioactive phosphorus ( 32 P) in phage DN Radioactive DN Pellet entrifuge Pellet Radioactivity (phage DN) found in pellet Figure Experiment Batch 1: Radioactive sulfur ( 35 S) in phage protein Labeled phages infect cells. Radioactive protein gitation frees outside phage parts from cells. entrifuged cells form a pellet. Free phages and phage parts remain in liquid. Radioactivity (phage protein) found in liquid DN entrifuge Pellet Figure Experiment Batch 2: Radioactive phosphorus ( 32 P) in phage DN Labeled phages infect cells. Radioactive DN gitation frees outside phage parts from cells. entrifuged cells form a pellet. Free phages and phage parts remain in liquid. entrifuge Pellet Radioactivity (phage DN) found in pellet 6

7 dditional Evidence hat DN Is the enetic Material It was known that DN is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group In 1950, Erwin hargaff reported that DN composition varies from one species to the next his evidence of diversity made DN a more credible candidate for the genetic material nimation: DN and RN Structure Figure 13.6 Sugarphosphate backbone end Nitrogenous bases hymine () denine () ytosine () end DN nucleotide uanine () 7

8 Figure Phosphate end DN nucleotide Sugar (deoxyribose) Nitrogenous base wo findings became known as hargaff s rules he base composition of DN varies between species In any species the percentages of and bases are equal and the percentages of and bases are equal he basis for these rules was not understood until the discovery of the double helix Building a Structural Model of DN: Scientific Inquiry James Watson and Francis rick were first to determine the structure of DN Maurice Wilkins and Rosalind Franklin were using a technique called X-ray crystallography to study molecular structure Franklin produced a picture of the DN molecule using this technique 8

9 Figure 13.7 (a) Rosalind Franklin (b) Franklin s X-ray diffraction photograph of DN Figure (a) Rosalind Franklin Figure (b) Franklin s X-ray diffraction photograph of DN 9

10 Franklin s X-ray crystallographic images of DN enabled Watson to deduce that DN was helical he X-ray images also enabled Watson to deduce the width of the helix and the spacing of the nitrogenous bases he pattern in the photo suggested that the DN molecule was made up of two strands, forming a double helix nimation: DN Double Helix Video: DN Surface Model 10

11 Figure 13.8 end Hydrogen bond end 3.4 nm 1 nm end 0.34 nm end (a) Key features of DN structure (b) Partial chemical structure (c) Space-filling model Figure nm 1 nm (a) Key features of DN structure 0.34 nm Figure end Hydrogen bond end end (b) Partial chemical structure end 11

12 Figure (c) Space-filling model Watson and rick built models of a double helix to conform to the X-ray measurements and the chemistry of DN Franklin had concluded that there were two outer sugar-phosphate backbones, with the nitrogenous bases paired in the molecule s interior Watson built a model in which the backbones were antiparallel (their subunits run in opposite directions) t first, Watson and rick thought the bases paired like with like ( with, and so on), but such pairings did not result in a uniform width Instead, pairing a purine with a pyrimidine resulted in a uniform width consistent with the X-ray data 12

13 Figure 13.9 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data Watson and rick reasoned that the pairing was more specific, dictated by the base structures hey determined that adenine () paired only with thymine (), and guanine () paired only with cytosine () he Watson-rick model explains hargaff s rules: in any organism the amount of = the amount of, and the amount of = the amount of Figure denine () hymine () uanine () ytosine () 13

14 oncept 13.2: Many proteins work together in DN replication and repair he relationship between structure and function is manifest in the double helix Watson and rick noted that the specific base pairing suggested a possible copying mechanism for genetic material Figure s1 (a) Parental molecule Figure s2 (a) Parental molecule (b) Separation of parental strands into templates 14

15 Figure s3 (a) Parental molecule (b) Separation of parental strands into templates (c) Formation of new strands complementary to template strands he Basic Principle: Base Pairing to a emplate Strand Since the two strands of DN are complementary, each strand acts as a template for building a new strand in replication In DN replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules Watson and rick s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or conserved from the parent molecule) and one newly made strand ompeting models were the conservative model (the two parent strands rejoin) and the dispersive model (each strand is a mix of old and new) 15

16 Figure Parent cell (a) onservative model First replication Second replication (b) Semiconservative model (c) Dispersive model Experiments by Matthew Meselson and Franklin Stahl supported the semiconservative model Figure Experiment Bacteria cultured in medium with 15 N (heavy isotope) Bacteria transferred to medium with 14 N (lighter isotope) Results DN sample centrifuged after first replication DN sample centrifuged after second replication Less dense More dense onclusion Predictions: First replication Second replication onservative model Semiconservative model Dispersive model 16

17 Figure Experiment Bacteria cultured in medium with 15 N (heavy isotope) Bacteria transferred to medium with 14 N (lighter isotope) Results DN sample centrifuged after first replication DN sample centrifuged after second replication Less dense More dense Figure onclusion Predictions: First replication Second replication onservative model Semiconservative model Dispersive model DN Replication: loser Look he copying of DN is remarkable in its speed and accuracy More than a dozen enzymes and other proteins participate in DN replication Much more is known about how this replication machine works in bacteria than in eukaryotes Most of the process is similar between prokaryotes and eukaryotes 17

18 etting Started Replication begins at sites called origins of replication, where the two DN strands are separated, opening up a replication bubble t each end of a bubble is a replication fork, a Y-shaped region where the parental strands of DN are being unwound nimation: DN Replication Overview nimation: Origins of Replication 18

19 Figure Primase opoisomerase Replication fork RN primer Helicase Single-strand binding proteins Helicases are enzymes that untwist the double helix at the replication forks Single-strand binding proteins bind to and stabilize single-stranded DN opoisomerase relieves the strain caused by tight twisting ahead of the replication fork by breaking, swiveling, and rejoining DN strands Figure (a) Origin of replication in an E. coli cell Origin of replication Doublestranded DN molecule Parental (template) strand Replication bubble Daughter (new) strand Replication fork wo daughter DN molecules 0.5 mm 19

20 Figure a 0.5 mm Multiple replication bubbles form and eventually fuse, speeding up the copying of DN Figure (b) Origins of replication in a eukaryotic cell Origin of replication Parental (template) strand Double-stranded DN molecule Daughter (new) strand Bubble Replication fork wo daughter DN molecules 0.25 mm 20

21 Figure a 0.25 mm Synthesizing a New DN Strand DN polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to an already existing chain base-paired with the template he initial nucleotide strand is a short RN primer he enzyme, primase, starts an RN chain with a single RN nucleotide and adds RN nucleotides one at a time using the parental DN as a template he primer is short (5 10 nucleotides long) he new DN strand will start from the end of the RN primer 21

22 Enzymes called DN polymerases catalyze the elongation of new DN at a replication fork Most DN polymerases require a primer and a DN template strand he rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells Each nucleotide that is added to a growing DN consists of a sugar attached to a base and to three phosphate groups dp is used to make DN and is similar to the P of energy metabolism he difference is in the sugars: dp has deoxyribose, while P has ribose s each monomer nucleotide joins the DN strand, it loses two phosphate groups as a molecule of pyrophosphate Figure New strand emplate strand Sugar Phosphate Base DN polymerase Nucleotide P P i Pyrophosphate 2 P i 22

23 ntiparallel Elongation Newly replicated DN strands must be formed antiparallel to the template strand DN polymerases add nucleotides only to the free end of a growing strand; therefore, a new DN strand can elongate only in the to direction long one template strand of DN, the DN polymerase synthesizes a leading strand continuously, moving toward the replication fork nimation: Leading Strand 23

24 Figure Leading strand Overview Origin of replication Lagging strand Primer Parental DN Lagging strand Overall directions of replication Leading strand Origin of replication RN primer Sliding clamp DN pol III ontinuous elongation in the to direction Figure Leading strand Overview Origin of replication Lagging strand Primer Lagging strand Overall directions of replication Leading strand Figure Parental DN Origin of replication RN primer Sliding clamp DN pol III ontinuous elongation in the to direction 24

25 o elongate the other new strand, the lagging strand, DN polymerase must work in the direction away from the replication fork he lagging strand is synthesized as a series of segments called Okazaki fragments fter formation of Okazaki fragments, DN polymerase I removes the RN primers and replaces the nucleotides with DN he remaining gaps are joined together by DN ligase nimation: Lagging Strand 25

26 nimation: DN Replication Review Figure Leading strand Overview Lagging Origin of replication strand Lagging strand emplate strand Overall directions of replication Primase makes RN primer. Origin of RN primer for fragment 1 DN pol III detaches. Leading strand Primer for leading replication strand DN pol III makes Okazaki fragment 1. Okazaki fragment 1 RN primer for fragment 2 Okazaki fragment 2 DN ligase forms bonds between DN fragments. DN pol III makes Okazaki fragment 2. DN pol I replaces RN with DN. Overall direction of replication Figure Leading strand Overview Lagging Origin of replication strand Lagging strand Overall directions of replication Leading strand 26

27 Figure s1 emplate strand Primase makes RN primer. Origin of replication Primer for leading strand Figure s2 emplate strand Primase makes RN primer. Origin of replication Primer for leading strand RN primer for fragment 1 DN pol III makes Okazaki fragment 1. Figure s3 emplate strand Primase makes RN primer. Origin of replication Primer for leading strand RN primer for fragment 1 DN pol III detaches. DN pol III makes Okazaki fragment 1. Okazaki fragment 1 27

28 Figure s1 RN primer for fragment 2 Okazaki fragment 2 DN pol III makes Okazaki fragment 2. Figure s2 RN primer for fragment 2 Okazaki fragment 2 DN pol III makes Okazaki fragment 2. DN pol I replaces RN with DN. Figure s3 RN primer for fragment 2 Okazaki fragment 2 DN pol III makes Okazaki fragment 2. DN ligase forms bonds between DN fragments. DN pol I replaces RN with DN. Overall direction of replication 28

29 he DN Replication omplex he proteins that participate in DN replication form a large complex, a DN replication machine he DN replication machine may be stationary during the replication process Recent studies support a model in which two DN polymerase molecules reel in parental DN and extrude newly made daughter DN molecules nimation: DN Replication Figure Leading strand template Parental DN onnecting protein DN pol III DN pol III Helicase Leading strand Lagging strand template Lagging strand Overall direction of replication 29

30 Proofreading and Repairing DN DN polymerases proofread newly made DN, replacing any incorrect nucleotides In mismatch repair of DN, other enzymes correct errors in base pairing hereditary defect in one such enzyme is associated with a form of colon cancer his defect allows cancer-causing errors to accumulate in DN faster than normal DN can be damaged by exposure to harmful chemical or physical agents such as cigarette smoke and X-rays It can also undergo spontaneous changes In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DN Figure s1 Nuclease 30

31 Figure s2 Nuclease DN polymerase Figure s3 Nuclease DN polymerase DN ligase Evolutionary Significance of ltered DN Nucleotides he error rate after proofreading repair is low but not zero Sequence changes may become permanent and can be passed on to the next generation hese changes (mutations) are the source of the genetic variation upon which natural selection operates 31

32 Replicating the Ends of DN Molecules For linear DN, the usual replication machinery cannot complete the ends of daughter strands Repeated rounds of replication produce shorter DN molecules with uneven ends Eukaryotic chromosomal DN molecules have special nucleotide sequences at their ends called telomeres Figure mm elomeres typically consist of multiple repetitions of one short nucleotide sequence elomeres do not prevent the shortening of DN molecules, but they do postpone it It has been proposed that the shortening of telomeres is connected to aging 32

33 If chromosomes of germ cells became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce n enzyme called telomerase catalyzes the lengthening of telomeres in germ cells elomerase is not active in most human somatic cells However, it does show inappropriate activity in some cancer cells elomerase is currently under study as a target for cancer therapies oncept 13.3: chromosome consists of a DN molecule packed together with proteins he bacterial chromosome is a double-stranded, circular DN molecule associated with a small amount of protein Eukaryotic chromosomes have linear DN molecules associated with a large amount of protein In a bacterium, the DN is supercoiled and found in a region of the cell called the nucleoid 33

34 hromatin, a complex of DN and protein, is found in the nucleus of eukaryotic cells hromosomes fit into the nucleus through an elaborate, multilevel system of packing hromatin undergoes striking changes in the degree of packing during the course of the cell cycle Proteins called histones are responsible for the first level of DN packing in chromatin Four types of histones are most common in chromatin: H2, H2B, H3, and H4 nucleosome consists of DN wound twice around a protein core of eight histones, two of each of the main histone types Figure DN double helix (2 nm in diameter) Nucleosome (10 nm in diameter) Histone tail Histones H1 30-nm fiber Looped domain Scaffold 300-nm fiber hromatid (700 nm) Replicated chromosome (1,400 nm) 34

35 Figure DN double helix (2 nm in diameter) Nucleosome (10 nm in diameter) Histones Histone tail H1 Figure a DN double helix (2 nm in diameter) Figure b Nucleosome (10 nm in diameter) 35

36 Figure hromatid (700 nm) 30-nm fiber Looped domain Scaffold 300-nm fiber Replicated chromosome (1,400 nm) Figure a 30-nm fiber Figure b Looped domain Scaffold 36

37 Figure c hromatid (700 nm) nimation: DN Packing Video: Nucleosome Model 37

38 t interphase, most of the chromatin is compacted into a 30-nm fiber, which is folded further in some areas by looping Even during interphase, centromeres and some other parts of chromosomes are highly condensed, similar to metaphase chromosomes his condensed chromatin is called heterochromatin; the more dispersed, less compacted chromatin is called euchromatin Dense packing of the heterochromatin makes it largely inaccessible to the machinery responsible for transcribing genetic information hromosomes are dynamic in structure; a condensed region may be loosened or modified as needed for various cell processes For example, histones can undergo chemical modifications that result in changes in chromatin organization oncept 13.4: Understanding DN structure and replication makes genetic engineering possible omplementary base pairing of DN is the basis for nucleic acid hybridization, the base pairing of one strand of a nucleic acid to another, complementary sequence Nucleic acid hybridization forms the foundation of virtually every technique used in genetic engineering, the direct manipulation of genes for practical purposes 38

39 DN loning: Making Multiple opies of a ene or Other DN Segment o work directly with specific genes, scientists prepare well-defined segments of DN in identical copies, a process called DN cloning Most methods for cloning pieces of DN in the laboratory share general features Many bacteria contain plasmids, small circular DN molecules that replicate separately from the bacterial chromosome o clone pieces of DN, researchers first obtain a plasmid and insert DN from another source ( foreign DN ) into it he resulting plasmid is called recombinant DN Figure Bacterium Bacterial chromosome Plasmid Recombinant DN (plasmid) ene inserted into plasmid (a cloning vector) ene of interest Plasmid put into bacterial cell ell containing gene of interest DN of chromosome ( foreign DN) Recombinant bacterium ene of interest opies of gene Host cell grown in culture to form a clone of cells containing the cloned gene of interest Protein expressed from gene of interest Protein harvested ene for pest resistance inserted into plants Basic research and various applications Human growth hormone treats stunted growth ene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy 39

40 Figure Bacterium Bacterial chromosome Plasmid Recombinant DN (plasmid) ene inserted into plasmid (a cloning vector) ene of interest Plasmid put into bacterial cell ell containing gene of interest DN of chromosome ( foreign DN) Recombinant bacterium ene of interest Host cell grown in culture to form a clone of cells containing the cloned gene of interest Protein expressed from gene of interest Figure ene of interest opies of gene Protein expressed from gene of interest Protein harvested Basic research and various applications ene for pest resistance inserted into plants Human growth hormone treats stunted growth ene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy he production of multiple copies of a single gene is called gene cloning he plasmid that carries the cloned DN is called a cloning vector ene cloning is used to make many copies of a gene and to produce a protein product he ability to amplify many copies of a gene is crucial for applications involving a single gene 40

41 Using Restriction Enzymes to Make Recombinant DN Bacterial restriction enzymes cut DN molecules at specific DN sequences called restriction sites restriction enzyme usually makes many cuts, yielding restriction fragments nimation: Restriction Enzymes Figure Bacterial plasmid DN Restriction enzyme cuts the sugar-phosphate backbones at each arrow. Restriction site DN fragment from another source is added. Base pairing of sticky ends produces various combinations. DN ligase seals the strands.. Sticky end One possible combination Fragment from different DN molecule cut by the same restriction enzyme Recombinant DN molecule Recombinant plasmid 41

42 Figure Bacterial plasmid DN Restriction enzyme cuts the sugar-phosphate backbones at each arrow. Restriction site Sticky end Figure DN fragment from another source is added. Base pairing of sticky ends produces various combinations. Sticky end Fragment from different DN molecule cut by the same restriction enzyme One possible combination Figure One possible combination DN ligase seals the strands. Recombinant DN molecule Recombinant plasmid 42

43 he most useful restriction enzymes cleave the DN in a staggered manner to produce sticky ends Sticky ends can bond with complementary sticky ends of other fragments DN ligase can close the sugar-phosphate backbones of DN strands o see the fragments produced by cutting DN molecules with restriction enzymes, researchers use gel electrophoresis his technique separates a mixture of nucleic acid fragments based on length Figure Mixture of DN molecules of different lengths Power source athode Wells node el (a) Negatively charged DN molecules will move toward the positive electrode. Restriction fragments of known lengths (b) Shorter molecules are slowed down less than longer ones, so they move faster through the gel. 43

44 Figure Mixture of DN molecules of different lengths athode Wells Power source node el (a) Negatively charged DN molecules will move toward the positive electrode. Figure Restriction fragments of known lengths (b) Shorter molecules are slowed down less than longer ones, so they move faster through the gel. mplifying DN in Vitro: he Polymerase hain Reaction (PR) and Its Use in loning he polymerase chain reaction (PR) can produce many copies of a specific target segment of DN three-step cycle brings about a chain reaction that produces an exponentially growing population of identical DN molecules he key to PR is an unusual, heat-stable DN polymerase called aq polymerase. 44

45 Figure echnique arget sequence enomic DN Denaturation nnealing ycle 1 yields 2 molecules Primers Extension New nucleotides ycle 2 yields 4 molecules ycle 3 2 of the 8 molecules (in white boxes) match target sequence and are the right length Figure echnique enomic DN arget sequence Figure s1 Denaturation ycle 1 yields 2 molecules 45

46 Figure s2 Denaturation ycle 1 yields 2 molecules nnealing Primers Figure s3 Denaturation ycle 1 yields 2 molecules nnealing Primers Extension New nucleotides Figure ycle 2 yields 4 molecules ycle 3 2 of the 8 molecules (in white boxes) match target sequence and are the right length Results fter 30 more cycles, over 1 billion (10 9 ) molecules match the target sequence. 46

47 PR amplification alone cannot substitute for gene cloning in cells Instead, PR is used to provide the specific DN fragment to be cloned PR primers are synthesized to include a restriction site that matches the site in the cloning vector he fragment and vector are cut and ligated together Figure loning vector (bacterial plasmid) DN fragment obtained by PR (cut by same restriction enzyme used on cloning vector) Mix and ligate Recombinant DN plasmid DN Sequencing Once a gene is cloned, complementary base pairing can be exploited to determine the gene s complete nucleotide sequence his process is called DN sequencing 47

48 Next-generation sequencing techniques, developed in the last 15 years, are rapid and inexpensive hey sequence by synthesizing the complementary strand of a single, immobilized template strand chemical technique enables electronic monitors to identify which nucleotide is being added at each step Figure (a) Next-generation sequencing machines 4-mer 3-mer 2-mer 1-mer (b) flow-gram from a next-generation sequencing machine Figure (a) Next-generation sequencing machines 48

49 Figure mer 3-mer 2-mer 1-mer (b) flow-gram from a next-generation sequencing machine Next-generation methods are being complemented or replaced by third-generation methods hese newer techniques are faster and less expensive Several groups are working on nanopore methods, which involve moving a single DN strand through a tiny pore in a membrane Nucleotides are identified by slight differences in the amount of time that they interrupt an electrical current across the pore Figure

50 Editing enes and enomes Over the past five years, biologists have developed a powerful new technique called the RISPR-as9 system as9 is a nuclease that cuts double-stranded DN molecules as directed by a guide RN that is complementary to the target gene Researchers have used this system to knock out (disable) a given gene in order to determine its function Researchers have also modified the RISPR-as9 system to repair a gene that has a mutation In 2014 a group of researchers reported using this system to successfully correct a mutated gene in mice RISPR technology is sparking widespread excitement among researchers and physicians Figure as9 active sites NULEUS uide RN complementary sequence as9 protein uide RN engineered to guide the as9 protein to a target gene omplementary sequence that can ctive sites that bind to a target gene can cut DN as9-guide RN complex Part of the target gene Resulting cut in target gene Normal (functional) gene for use as a template by repair enzymes YOPLSM (a) Scientists can disable (b) If the target gene has a ( knock out ) the target gene mutation, it can be repaired. to study its normal function. NULEUS Random nucleotides Normal nucleotides 50

51 Figure as9 protein uide RN engineered to guide the as9 protein to a target gene ctive sites that can cut DN as9-guide RN complex omplementary sequence that can bind to a target gene Figure YOPLSM as9 active sites uide RN complementary sequence NULEUS Part of the target gene Resulting cut in target gene Figure (a) Scientists can disable ( knock out ) the target gene to study its normal function. Normal (functional) gene for use as a template by repair enzymes (b) If the target gene has a mutation, it can be repaired. Random nucleotides Normal nucleotides 51

52 Figure (a) Origin of replication in an E. coli cell Origin of replication Doublestranded DN molecule wo daughter DN molecules Parental (template) strand Replication bubble Daughter (new) strand Replication fork (b) Origins of replication in a eukaryotic cell Origin of replication Parental (template) strand Bubble Double-stranded DN molecule Daughter (new) strand Replication fork wo daughter DN molecules 0.5 mm 0.25 mm Figure Helicase Parental DN Single-strand binding proteins Lagging strand template Leading strand template Leading strand Overview Origin of replication Leading strand Lagging strand Leading Lagging strand strand Overall directions of replication DN pol III Primer Primase Lagging strand DN pol III DN pol I DN ligase Figure Overview Origin of replication Leading strand Lagging strand Lagging strand Overall directions of replication Leading strand 52

53 Figure Single-strand binding proteins Leading strand template Helicase Leading strand Parental DN DN pol III Primer Primase Lagging strand template Figure Lagging strand DN pol III DN pol I DN ligase Lagging strand template Figure 13.UN

54 Figure 13.UN01-2 Sea urchin Figure 13.UN02 Nitrogenous bases Sugar-phosphate backbone Hydrogen bond Figure 13.UN03 DN pol III synthesizes leading strand continuously Parental DN Helicase DN pol III starts DN synthesis at end of primer, continues in direction Lagging strand synthesized in short Okazaki fragments, later joined by DN ligase Origin of replication Primase synthesizes a short RN primer DN pol I replaces the RN primer with DN nucleotides 54

55 Figure 13.UN04 Sticky end Figure 13.UN05 Figure 13.UN06 55