DNA Replication - BIOL 360 Module 2 PDF

Kwame Nkrumah University of Science & Technology, Kumasi, Ghana Module 2 DNA Replication Prof. Matthew Glover Addo Start Module  Featured image Module 2 Module Dashboard...

Kwame Nkrumah University of Science & Technology, Kumasi, Ghana Module 2 DNA Replication Prof. Matthew Glover Addo Start Module  Featured image Module 2 Module Dashboard FILTER DNA Replication Lessons Assignments 4 Start Learning 3 Dashboard hig Pick a Lesson Introduction Lesson 1 Method of t1 Lessons h1 title Replication Assignments Lesson 2 t2 Mechanism of Play h2 Replication Watch an overview Lesson 3 video on DNA t3 Rate of Replication h3 2  Featured image FILTER Module 2 Module Objectives DNA Replication Dashboard 1 Consider the modes of replication i.e. dispersive, conservative and semiconservative Introduction hig Lessons 2 Consider the experiments of Meselson and Stahl title Assignments Their experiment gave a convincing evidence to support the semiconservative nature of replication 3  Featured image FILTER Module 2 Module Objectives DNA Replication Dashboard 3 To appreciate how replication is initiated, elongated and terminated Introduction Emphasis will be laid on some of the enzymes hig Lessons involved in these processes title Assignments 4 DNA repair mechanism 4  Featured image FILTER Module 2 Module Lessons DNA Replication Dashboard Introduction t1 t2 t3 Lessons hig Lesson 1 h1 Lesson 2 title h2 Lesson 3 h3 Assignments Method of Mechanism of Rate of Replication Replication Replication Start Start Start 5  Module 2 DNA Replication t1 h1 Lesson 1 hig Method of DNA Replication t3 Learn about the three hypotheses that were previously proposed for the method of replication of DNA t2 Learn Now  Lesson 1 Method of DNA Replication t1 FILTER To explain the phenomenon of heredity, biological information must hig be accurately h1 copied (replicated) and transmitted from each cell to all of its progeny 7 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses t1 FILTER Conservative Dispersive Semiconservative hig h1 1 2 3 8 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Conservative t1 FILTER Conservative replication would leave intact the original DNA molecule and generate a hig h1 1 completely new molecule 9 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Dispersive t1 FILTER Dispersive replication would produce two DNA molecules with sections of both old and hig 2 h1 new DNA interspersed along each strand 10 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Dispersive t1 FILTER The dispersive hypothesis is exemplified by a model proposed by Max Delbrück hig h1 2 Learn More 11 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Dispersive t1 FILTER ▪ He attempted to solve the problem of unwinding the two strands of the double helix by a mechanism that hig breaks the DNA backbone h1 2 every 10 nucleotides ▪ Untwist the molecule, and attaches the old strand to 12 t3 t2 the newly synthesized one  Lesson 1 Method of DNA Replication Proposed Hypotheses Dispersive t1 FILTER This would synthesize the DNA in short pieces alternating from one hig 2 h1 strand to the other 13 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Semiconservative replication would produce molecules with both old and new DNA, but each molecule would be hig 3 h1 composed of one old strand and one new one 14 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Each strand acts as a template or guide for the synthesis of a new hig DNA molecule by h1 3 the sequential addition of complementary base pairs 15 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Thereby generating a new DNA strand that is the complementary hig 3 h1 sequence to the parental DNA 16 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Each daughter DNA molecule ends up with hig one of the original h1 strands and one newly 3 synthesized strand 17 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Experiment In 1958 Matthew Meselson & Franklin Stahl worked out a clever hig procedure to distinguish h1 3 semi conservative DNA replication from conservative or dispersive replication, using a nonradioactive heavy isotope of nitrogen 18 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Experiment Meselson and Stahl opted for nitrogen hig because it is an h1 3 essential chemical component of DNA 19 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Experiment Therefore, every time a cell divides and its DNA replicates, it incorporates new N atoms into the DNA of either one or both of its two daughter cells, depending on which model was correct hig h1 3 20 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Experiment Ordinary nitrogen, the most abundant hig isotope, has an 3 atomic weight of 14, h1 so it is called 14N 21 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Experiment A relatively rare isotope 15N has hig an atomic weight 3 of 15 h1 22 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Experiment Meselson and Stahl found that if bacteria are grown in a medium enriched in 15N, they incorporate the heavy hig 3 h1 isotope into their DNA, which becomes denser than normal 23 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Experiment This labeled DNA clearly separates from ordinary DNA hig in gradient of h1 3 Cesium Chloride (CsCl) spun in an ultracentrifuge 24 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Experiment CsCl is used because it is a very dense salt and therefore makes dense enough solution that DNA hig 3 h1 will float somewhere in the middle rather than sinking to the bottom 25 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Experiment The aim of the experiment was to grow 15N labeled bacteria in 14N- hig 3 h1 medium and then to look at the density of the DNA products 26 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Experiment That is, E. coli cells with only 15N in their DNA were transferred to a 14N hig 3 h1 medium and were allowed to divide or replicate 27 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Experiment The progress of cell division was monitored by measuring the hig 3 h1 optical density of the cell suspension 28 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Results DNA was extracted periodically and was compared to pure 14N hig 3 h1 DNA and 15N DNA 29 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Results Based on these findings, the scientists were immediately able to exclude the conservative model of replication as a possibility hig h1 1 2 3 30 Conservative Dispersive Semiconservative t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Results After all, if DNA replicated conservatively, there should have been two distinct bands after a 1 hig h1 single round of replication 31 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Results However, this result was consistent with both semiconservative 2 3 hig h1 and dispersive replication Dispersive Semiconservative 32 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Experiment To differentiate between the two, Meselson and Stahl hig had to let the cells 3 divide again and then h1 sample the DNA after a second round of replication 33 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Results DNA from cells after two replications had been completed was hig found to consist of h1 3 equal amounts of DNA with two different densities 34 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Results Intermediate density of DNA of cells grown DNA from cells for only one division in grown exclusively 14N medium in 14N medium hig h1 3 3 35 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Results This was inconsistent with dispersive replication 3 2 After all, if the dispersive hig h1 model were the correct model, the scientists would have continued to observe only a single band after every round of replication 36 t3 t2 Dispersive Semiconservative  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Results Dispersive replication would have resulted in double-stranded DNA with both strands hig having mixtures of 15N h1 and 14N DNA, either of 2 which would have appeared as DNA of an intermediate density Dispersive 37 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Results Semiconservative replication would result in double- stranded DNA with 3 hig h1 one strand of 15N DNA, and one of 14N DNA Semiconservative 38 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Results This will result in equal hig amounts of DNA with h1 3 two different densities Semiconservative 39 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Results The scientists continued to observe the same two bands after several subsequent 3 hig h1 rounds of replication Semiconservative 40 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Results These results were consistent with the semiconservative model of replication and the hig reality that, when DNA h1 3 replicated, each new double helix was built with one old strand and one new strand Semiconservative 41 t3 t2  Lesson 1 Method of DNA Replication Proposed Hypotheses Semiconservative t1 FILTER Results The result was consistent with the semiconservative replication hypothesis hig 3 h1 1 2 42 Conservative Dispersive Semiconservative t3 t2  Lesson 1 Method of DNA Replication Replication of Eukaryotic Chromosomes t1 FILTER Before a cell can divide, it must hig duplicate or h1 replicate all of its DNA 43 t3 t2  Lesson 1 Method of DNA Replication Replication of Eukaryotic Chromosomes t1 FILTER In eukaryotes, G2 Cell Division this occurs S M hig during S phase of h1 Interphase g2 g1 mi mcsa p it the cell cycle G1 44 t3 t2  Lesson 1 Method of DNA Replication Replication of Eukaryotic Chromosomes t1 FILTER Eukaryotic DNA replication is very slow compared hig to E. coli DNA replication: only h1 about 75 nucleotides/ second 45 t3 t2  Featured image FILTER Module 2 Module Lessons DNA Replication Dashboard Introduction t1 t2 t3 Lessons hig Lesson 1 h1 Lesson 2 title h2 Lesson 3 h3 Assignments Method of Mechanism of Rate of Replication Replication Replication Start Start Start 46  Module 2 DNA Replication Lesson 2 t2 h2 hig Mechanism of DNA Replication t3 Learn about the mechanism DNA uses to replicate itself to form new DNAs t1 Learn Now 47  Lesson 2 Mechanism of DNA Replication t2 FILTER Replication is a huge task, whether in bacteria or in hig eukaryotes and h2 requires many proteins or enzymes to act together 48 t3 t1  Lesson 2 Mechanism of DNA Replication Physical & Biochemical Challenges Cells Overcome t2 FILTER 1 Locating site(s) to begin replication The proper enzymes must be collected or deposited there hig h2 49 t3 t1  Lesson 2 Mechanism of DNA Replication Physical & Biochemical Challenges Cells Overcome t2 FILTER 1 Locating site(s) to begin replication 2 Unwinding the hig double helix to expose the two h2 strands. This imposes twisting strain on the portions of the helix farther away from the 50 t3 t1 unwinding site  Lesson 2 Mechanism of DNA Replication Physical & Biochemical Challenges Cells Overcome t2 FILTER 1 Locating site(s) to begin replication 2 Unwinding the double helix hig This is much like how untangling a twisted h2 phone cord does, and those forces must be relieved to prevent breakage of 51 t3 t1 the DNA strands  Lesson 2 Mechanism of DNA Replication Origins t2 FILTER DNA replication begins at specific points known as the Origins hig These sites are h2 recognized by certain proteins in the cell 52 t3 t1  Lesson 2 Mechanism of DNA Replication Origins t2 FILTER The Origins are rich in Adenine and Thymine bases, as breaking 2 hydrogen bonds between the hig Adenine and Thymine is easier h2 than breaking the triple hydrogen bonds between Cytosine and Guanine 53 t3 t1  Lesson 2 Mechanism of DNA Replication Replication Fork t2 FILTER 6 proteins arranged in a ring shape known as Helicase, unwind the double stranded DNA helix into single hig h2 strands by breaking the hydrogen bonds between them 54 t3 t1  Lesson 2 Mechanism of DNA Replication Replication Fork t2 FILTER This results in the formation of a replication fork hig The replication fork is a h2 structure that is formed during the DNA replication process 55 t3 t1  Lesson 2 Mechanism of DNA Replication Replication Fork t2 FILTER The fork is made with the action of helicase, that breaks the hydrogen bonds, that hig h2 hold the two DNA strands together 56 t3 t1  Lesson 2 Mechanism of DNA Replication Replication Fork t2 FILTER This results in a structure that has hig two branching h2 'prongs' of a single strand DNA each 57 t3 t1  Lesson 2 Mechanism of DNA Replication Tetramers t2 FILTER Tetramers, known as the single stranded hig binding proteins, h2 cover the single- stranded DNA 58 t3 t1  Lesson 2 Mechanism of DNA Replication Tetramers t2 FILTER This prevents the DNA strands from hig re-annealing and h2 forming the double stranded molecule 59 t3 t1  Lesson 2 Mechanism of DNA Replication The two single DNA strands t2 FILTER The two single DNA strands act as templates hig individually, that h2 are used for producing two complementary DNA strands 60 t3 t1  Lesson 2 Mechanism of DNA Replication The two single DNA strands t2 FILTER The double helix consists of two anti-parallel DNA strands with hig h2 complementary 5' to 3' strands 61 t3 t1  Lesson 2 Mechanism of DNA Replication Initiating the Replication Processt2 FILTER An RNA polymerase known as primase, synthesizes short hig RNA primers (about h2 60nt long) that initiate the DNA replication process 62 t3 t1  Lesson 2 Mechanism of DNA Replication Initiating the Replication Processt2 FILTER An enzyme called DNA polymerase binds to one hig strand of the DNA and begins h2 moving along it in the 3' to 5' direction 63 t3 t1  Lesson 2 Mechanism of DNA Replication Initiating the Replication Processt2 FILTER DNA polymerase cannot begin synthesizing the DNA hig strand initially h2 It needs a nucleic acid chain in the beginning to begin copying the strand 64 t3 t1  Lesson 2 Mechanism of DNA Replication Initiating the Replication Processt2 FILTER The original DNA strand is used as a template to synthesize the DNA strand in the 5' → 3' hig h2 direction with the help of an extension formed by RNA primer 65 t3 t1  Lesson 2 Mechanism of DNA Replication Initiating the Replication Processt2 FILTER DNA polymerase hig can synthesize the h2 strand in 5' → 3' direction only 66 t3 t1  Lesson 2 Mechanism of DNA Replication Initiating the Replication Processt2 FILTER It hooks the 5' phosphate group of an incoming nucleotide onto the 3' hig hydroxyl group at the h2 end of the growing nucleic acid chain 67 t3 t1  Lesson 2 Mechanism of DNA Replication Initiating the Replication Processt2 FILTER In eukaryotes, this molecule is hig called DNA h2 polymerase delta (δ) 68 t3 t1  Lesson 2 Mechanism of DNA Replication Initiating the Replication Processt2 FILTER The DNA polymerase uses the strand as a template for assembling a hig h2 leading strand of nucleotides and reforming a double helix 69 t3 t1  Lesson 2 Mechanism of DNA Replication Synthesis of the Leading Strand t2 FILTER The DNA strand that is read in the 3' → 5' and synthesized in hig the 5' → 3' direction h2 continuously, is known as the leading strand 70 t3 t1  Lesson 2 Mechanism of DNA Replication Synthesis of the Leading Strand t2 FILTER DNA polymerase III synthesiSes the hig DNA using the 3'- h2 OH group, donated by the single RNA primer 71 t3 t1  Lesson 2 Mechanism of DNA Replication Synthesis of the Leading Strand t2 FILTER The DNA replication continues in the hig direction of the h2 replication fork, in a continuous manner 72 t3 t1  Lesson 2 Mechanism of DNA Replication Synthesis of the Leading Strand t2 FILTER RNAse H and DNA polymerase I (exonuclease) recognizes the RNA hig polymers that are h2 bound to the DNA template and removes the primers by RNA hydrolysis 73 t3 t1  t2 FILTER Module 2 Module Assignments DNA Replication Assignment 1 Dashboard Introduction Read on Lessons DNA POL I, POL II and POL III t h2 i t l e Assignments hig 74 t3 t1  Lesson 2 Mechanism of DNA Replication Synthesis of the Lagging Strand t2 FILTER The lagging strand is the DNA strand of hig the replication fork, h2 that is opposite to the leading strand 75 t3 t1  Lesson 2 Mechanism of DNA Replication Synthesis of the Lagging Strand t2 FILTER Because DNA synthesis can only occur in 5' to 3‘ direction, a molecule of a second type of DNA hig polymerase (epsilon, ε, h2 in eukaryotes) binds to the other template strand as the double helix opens 76 t3 t1  Lesson 2 Mechanism of DNA Replication Synthesis of the Lagging Strand t2 FILTER This molecule synthesize discontinuous hig segments of h2 polynucleotides (called Okazaki fragments) 77 t3 t1  Lesson 2 Mechanism of DNA Replication Synthesis of the Lagging Strand t2 FILTER Primase builds RNA primers in short bursts over the lagging strand, which is hig h2 synthesized in the 5' → 3' by DNA polymerase 78 t3 t1  Lesson 2 Mechanism of DNA Replication Synthesis of the Lagging Strand t2 FILTER The RNA primers are then removed and new deoxyribonucleotides are added to the gaps, hig h2 where the RNA was present 79 t3 t1  Lesson 2 Mechanism of DNA Replication Synthesis of the Lagging Strand t2 FILTER DNA Polymerase continues with the synthesis of the new hig h2 DNA strand 80 t3 t1  Lesson 2 Mechanism of DNA Replication Synthesis of the Lagging Strand t2 FILTER Finally, DNA ligase (an enzyme) joins the hig deoxyribonucleotides together, thus h2 completing the lagging strand 81 t3 t1  Lesson 2 Mechanism of DNA Replication Termination of Replication t2 FILTER In eukaryotes, termination of replication is hig h2 poorly understood 82 t3 t1  Lesson 2 Mechanism of DNA Replication Termination of Replication t2 FILTER Eukaryotes have linear DNA, and therefore use hig telomeres, which are h2 GT rich repeating units that 'protect' the end of the DNA 83 t3 t1  Featured image FILTER Module 2 Module Lessons DNA Replication Dashboard Introduction t1 t2 t3 Lessons hig Lesson 1 h1 Lesson 2 title h2 Lesson 3 h3 Assignments Method of Mechanism of Rate of Replication Replication Replication Start Start Start 84  Module 2 DNA Replication t3 h3 hig Lesson 3 Rate of Replication t2 Find out how long it takes for DNA to replicate t1 Learn Now  Lesson 3 Rate of Replication t3 FILTER The single molecule of DNA, that is the hig E. coli genome h3 contains 4.7 x 106 nucleotide pairs 86 t2 t1  Lesson 3 Rate of Replication t3 FILTER DNA replication begins at a single, fixed location in this molecule, called the hig replication origin, proceeds at about h3 1000 nucleotides per second, and thus is done in no more than 40 minutes 87 t2 t1  Lesson 3 Rate of Replication t3 FILTER Due to the precision of the process (which includes a "proof- reading" function), hig the job is done with h3 only about one incorrect nucleotide for every 109 nucleotides inserted 88 t2 t1  Lesson 3 Rate of Replication t3 FILTER In other words, more often than hig not, the E. coli h3 genome (4.7 x 106) is copied without error! 89 t2 t1  Lesson 3 Rate of Replication t3 FILTER The Genome of complex hig eukaryotes is huge h3 as compared to procaryotes 90 t2 t1  Lesson 3 Rate of Replication t3 FILTER The speed of DNA replication for hig human is about h3 50-75 nucleotides per second per replication fork 91 t2 t1  Lesson 3 Rate of Replication t3 FILTER However, the genome can be copied only in a few hours because many replication hig h3 forks take place at the same time (multiple initiation sites) 92 t2 t1  Featured image FILTER Module 2 Module Lessons DNA Replication Dashboard Introduction t1 t2 t3 Lessons hig Lesson 1 h1 Lesson 2 title h2 Lesson 3 h3 Assignments Method of Mechanism of Rate of Replication Replication Replication Start Start Start 93  Module 2 DNA Replication hig t3 End of 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