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CHAPTER 3 DNA Replication

CHAPTER 3 DNA Replication Peter J. Russell edited by Yue-Wen Wang Ph. D. Dept. of Agronomy, NTU 1. Watson and Crick DNA model implies a mechanism for replication: a. Unwind the DNA molecule. b. Separate the two strands. c. Make a complementary copy for each strand.

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CHAPTER 3 DNA Replication

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  1. CHAPTER 3DNA Replication Peter J. Russell edited by Yue-Wen Wang Ph. D.Dept. of Agronomy, NTU 台大農藝系 遺傳學 601 20000

  2. 1. Watson and Crick DNA model implies a mechanism for replication: a. Unwind the DNA molecule. b. Separate the two strands. c. Make a complementary copy for each strand. Semiconservative DNA Replication 台大農藝系 遺傳學 601 20000

  3. 2 .Three possible models were proposed for DNA replication: a. Conservative model proposed both strands of one copy would be entirely old DNA, while the other copy would have both strands of new DNA. b. Dispersive model was that dsDNA might fragment, replicate dsDNA, and then reassemble, creating a mosaic of old and new dsDNA regions in each new chromosome. c. Semiconservative model is that DNA strands separate, and a complementary strand is synthesized for each, so that sibling chromatids have one old and one new strand. This model was the winner in the Meselson and Stahl experiment. 台大農藝系 遺傳學 601 20000

  4. Fig. 3.1 Three models for the replication of DNA 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  5. Animation: The Meselson-Stahl Experiment 1. Meselson and Stahl (1958) grew E. coli in a heavy (not radioactive) isotope of nitrogen, 15N in the form of 15NH4Cl. Because it is heavier, DNA containing 15N is more dense than DNA with normal 14N, and so can be separated by CsCl density gradient centrifugation (Box 3.1). 2. Once the E. coli were labeled with heavy 15N, the researchers shifted the cells to medium containing normal 14N, and took samples at time points. DNA was extracted from each sample and analyzed in CsCl density gradients (Figure 3.2). The Meselson-Stahl Experiment 台大農藝系 遺傳學 601 20000

  6. Fig. 3.2 The Meselson-Stahl experiment, which showed that DNA replicates semiconservatively 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  7. Box Fig. 3.1 Equilibrium centrifugation of DNA of different densities in a cesium chloride density gradient 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  8. 3. After one replication cycle in normal 14N medium, all DNA had density intermediate between heavy and normal. After two replication cycles, there were two bands in the density gradient, one at the intermediate position, and one at the position for DNA containing entirely 14N. 4. Results compared with the three proposed models: a. Does not fit conservative model, because after one generation there is a single intermediate band, rather than one with entirely 15N DNA and another with entirely 14N DNA. b. The dispersive model predicted that a single band of DNA of intermediate density would be present in each generation, gradually becoming less dense as increasing amounts of 14N were incorporated with each round of replication. Instead, Meselson and Stahl observed two bands of DNA, with the intermediate form decreasing over time. c. The semiconservative model fits the data very well. 台大農藝系 遺傳學 601 20000

  9. 1. To visualize DNA of eukaryotic chromosomes replicating, CHO (Chinese hamster ovary) cells are grown in 5-bromodeoxyuridine (BUdR), a base analog for thymine. After two rounds of replication, mitotic chromosomes are stained with fluorescent dye and Giemsa stain (Figure 3.3). Semiconservative DNA Replication in Eukaryotes 台大農藝系 遺傳學 601 20000

  10. 2. DNA containing T stains darkly, while DNA containing two BUdR strands stains lightly. Observed that after one generation, both chromatids stain the same, each with one BUdR strand and one T strand. After two generations, they stain differently and are called harlequin chromosomes, one light (both strands have BUdR) and one dark (one strand has BUdR and other strand has T). 3. Showed that eukaryotes also use semiconservative DNA replication. 台大農藝系 遺傳學 601 20000

  11. 1. First isolation of an enzyme involved in DNA replication was in 1955. Arthur Kornberg won the 1959 Nobel Prize in Physiology or Medicine for this work. Enzymes Involved in DNA Synthesis 台大農藝系 遺傳學 601 20000

  12. 1. Accomplished in vitro synthesis of E. coli DNA. His reaction mixture included: a. DNA fragments (template). b. Radioactively labeled dNTPs (dATP, dGTP, dTTP and dCTP). c. E. coli lysate. 2. Enzyme originally called the Kornberg enzyme, now known as DNA Polymerase I. Once isolated, could characterize its activity, showing that the above components are required, along with Mg21 ions for maximum activity. DNA Polymerase I 台大農藝系 遺傳學 601 20000

  13. Animation: DNA Biosynthesis: How a New DNA Strand is Made 1. All DNA polymerases link dNTPs into DNA chains (Figure 3.4). Main features of the reaction: a. An incoming nucleotide is attached by its 5’-phosphate group to the 3’-OH of the growing DNA chain. Energy comes from the dNTP releasing two phosphates. The DNA chain acts as a primer for the reaction. b. The incoming nucleotide is selected by its ability to hydrogen bond with the complementary base in the template strand. The process is fast and accurate. c. DNA polymerases synthesize only from 5’ to 3’. 2. Two additional DNA polymerases were later isolated, DNA Pol II in 1970 and DNA Pol III in 1971. Roles of DNA Polymerases 台大農藝系 遺傳學 601 20000

  14. Fig. 3.4a DNA chain elongation catalyzed by DNA polymerase 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  15. Fig. 3.4b DNA chain elongation catalyzed by DNA polymerase 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  16. 3. The properties of these enzymes are (Table 3.1): a. DNA polymerase I is a single peptide encoded by polA. There are about 400 molecules in an E. coli cell. Replicates DNA in the 5’ →3’ direction. Has 5’ →3’ exonuclease activity to remove nucleotides from 5’ end of DNA or from RNA primer. b. DNA polymerase II is a single peptide encoded by polB. Number of molecules per E. coli cell isn’t known for certain, but probably around 10–20. Role in the cell is unknown. c. DNA polymerase III has 10 polypeptide subunits encoded by 10 different genes. The catalytic core of the enzyme is composed of three subunits, α (encoded by the dnaE gene), ε (dnaQ) and θ (holE). There are 10–20 molecules of this enzyme in an E. coli cell. Replicates DNA in the 5’ →3’ direction. 4. All three E. coli DNA polymerases have 3’ →5’ exonuclease (proofreading) activity. The properties of DNA polymerases 台大農藝系 遺傳學 601 20000

  17. 1. Table 3.2 shows key genes and DNA sequences involved in replication. Molecular Model of DNA Replication 台大農藝系 遺傳學 601 20000

  18. 1. Replication starts at origin of replication, with denaturation to expose the bases and create a bi-directional replication bubble. E. coli has one origin, oriC, which has a minimal sequence of about 245 bp required for initiation. 2. Events in initiating DNA synthesis, derived from in vitro studies (Figure 3.5): a. Gyrase (a type of topoisomerase) relaxes supercoils in the region. b. Initiator proteins attach. c. DNA helicase (from dnaB) binds initiator proteins on the DNA, and denatures the region using ATP as an energy source. d. DNA primase (from dnaG) binds helicase to form a primosome, which synthesizes a short (11 6 1 nt) RNA primer. Primers begin with two purines, typically AG. Initiation o Molecular Model of DNA Replication 台大農藝系 遺傳學 601 20000

  19. Fig. 3.5 Model for the formation of a replication bubble at a replication origin in E. coli and the initiation of the new DNA strand 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  20. Animation: Molecular Model of DNA Replication Semidiscontinuous DNA Replication 台大農藝系 遺傳學 601 20000

  21. Fig. 3.6a, b Model for the events occurring around a single replication fork of the E. coli chromosome 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  22. Fig. 3.6c-e Model for the events occurring around a single replication fork of the E. coli chromosome 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  23. 1. When DNA denatures at the oriC, replication forks are formed. DNA replication is usually bi-directional, but will consider events at just one replication fork (Figure 3.6): a. Single-strand DNA-binding proteins (SSBs) bind the ssDNA formed by helicase, preventing reannealing. b. Primase synthesizes a primer on each template strand. c. DNA polymerase III adds nucleotides to the 3’ end of the primer, synthesizing a new strand complementary to the template, and displacing the SSBs. DNA is made in opposite directions on the two template strands. d. New strand made 5’ → 3’ in same direction as movement of the replication fork is leading strand, while new strand made in opposite direction is lagging strand. Leading strand needs only one primer, while lagging needs a series of primers. 台大農藝系 遺傳學 601 20000

  24. 2. Helicase denaturing DNA causes tighter winding in other parts of the circular chromosome. Gyrase relieves this tension. 3. Leading strand is synthesized continuously, while lagging strand is synthesized discontinuously, in the form of Okazaki fragments. DNA replication is therefore semidiscontinuous. 4. Each fragment requires a primer to begin, and is extended by DNA polymerase III. 5. Okazaki data show that these fragments are gradually joined together to make a full-length dsDNA chromosome. DNA polymerase I uses the 3’-OH of the adjacent DNA fragment as a primer, and simultaneously removes the RNA primer while resynthesizing the primer region in the form of DNA. The nick remaining between the two fragments is sealed with DNA ligase. (Fig. 3.7) 台大農藝系 遺傳學 601 20000

  25. Fig. 3.7 Action of DNA ligase in sealing the gap between adjacent DNA fragments to form a longer, covalently continuous chain 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  26. 6. Key proteins are associated to form a replisome. Template DNA probably bends to allow synthesis of both leading and lagging strands at the replication fork (Fig. 3.8) 7. Early stages of bidirectional replication are summarized in Figure 3.9. 台大農藝系 遺傳學 601 20000

  27. Fig. 3.8 Model for the “replication machine,” or replisome, the complex of key replication proteins, with the DNA at the replication fork 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  28. iActivity: Unraveling DNA Replication 台大農藝系 遺傳學 601 20000

  29. Fig. 3.9 Diagram of the formation at a replication origin sequence of two replication forks that move in opposite directions 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  30. Fig. 3.10 Bidirectional replication of circular DNA molecules 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  31. 1. Some circular chromosomes (e.g., E. coli) are circular throughout replication, creating a theta-like (θ) shape (Fig. 3.10). As the strands separate on one side of the circle, positive supercoils form elsewhere in the molecule. Replication fork moves about 500 nt/ second, so at 10 bp/turn, replication fork rotates at 3,000 rpm. 2. Topoisomerases relieve the supercoils, allowing the DNA strands to continue separating as the replication forks advance. (Fig. 3.11) Replication of circular DNA and the supercoiling problem 台大農藝系 遺傳學 601 20000

  32. Fig. 3.11 Diagram showing the unreplicated, supercoiled parent strands and the portions already replicated 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  33. 1. Another model for replication is rolling circle, which is used by several bacteriophages, including ΦX174 (after a complement is made for the genomic ssDNA) and λ (after circularization by base pairing between the “sticky” ssDNA cos ends) 2. Rolling circle replication begins with a nick (single-stranded break) at the origin of replication. The 5’ end is displaced from the strand, and the 3’ end acts as a primer for DNA polymerase III, which synthesizes a continuous strand using the intact DNA molecule as a template. 3. The 5’ end continues to be displaced as the circle “rolls”, and is protected by SSBs until discontinuous DNA synthesis makes it a dsDNA again. Rolling Circle Replication 台大農藝系 遺傳學 601 20000

  34. Fig. 3.12 The replication process of double-stranded circular DNA molecules through the rolling circle mechanism 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  35. 4. A DNA molecule many genomes in length can be made by rolling circle replication. During viral assembly it is cut into individual viral chromosomes and packaged into phage head. 5. Bacteriophage λ, regardless of whether entering the lytic or lysogenic pathway, circularizes its chromosome immediately after infection. a. In a lysogenic infection, the circular DNA integrated into a specific site in the E. coli chromosome by a crossover event. b. In a lytic infection, rolling circle replication produces a long concatamer of λ DNA, and the a viral endonuclease (product of the ter gene) recognize the cos sites and makes the staggered cuts that used to assemble new virus particles. 台大農藝系 遺傳學 601 20000

  36. Fig. 3.13 Life cycle of a temperate phage, such as  台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  37. Fig. 3.14  chromosome structure varies at stages of lytic infection of E. coli 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  38. 1. DNA replication is very similar in both prokaryotes and eukaryotes, except that eukaryotes have more than one chromosome. DNA Replication in Eukaryotes 台大農藝系 遺傳學 601 20000

  39. 1. Each eukaryotic chromosome must be copied, so both DNA and histones must be doubled in each cell cycle. Chromosome duplication occurs during the S phase of the cell cycle, and segregation of progeny chromosomes during the M phase. 2. Details of cell cycle control are beyond the scope of this discussion. Yeasts, in which chromosomal replication is well studied, serve as a eukaryotic model organism. 3. Many genes are involved in the system of checks and balances that controls the cell cycle (Figure 3.15). Some major checkpoints in the system: a. To move from G1 into S phase, cells must be large enough, and the environment favorable. This checkpoint is called START in yeasts and G1 checkpoint in mammalian cells. b. The G2 checkpoint controls entry into M phase, assessing whether DNA has been duplicated, the cell is large enough and the environment is favorable. c. Attachment of chromosomes to the mitotic spindle during M phase functions as another checkpoint, triggering separation of the chromatids. DNA Replication and the Cell Cycle 台大農藝系 遺傳學 601 20000

  40. 4. Checkpoints use cyclin proteins and cyclin-dependent kinases (Cdks). Yeasts use a single Cdk for both the G1 and G2 checkpoints, while mammals use at least two Cdks for each checkpoint. In the yeast system, for example: a. At the START (G1) checkpoint, one or more G1 cyclins bind and activate the Cdk. The activated Cdk phosphorylates proteins needed to initiate S phase. Cyclin levels then decrease by proteolysis. b. At the G2 checkpoint, mitotic cyclin(s) bind to Cdk, forming the M-phase promoting factor (MPF). Dephosphorylation of MPF by other enzymes activates it, and it then catalyzes phosphorylation of proteins that move the cell into M phase. Mitotic cyclin is degraded after metaphase, causing MPF inactivation so that mitosis can be completed. 5. Cell cycle control is very complex, and will be discussed more fully with the genetics of cancer. 台大農藝系 遺傳學 601 20000

  41. Fig. 3.15 Some of the molecular events that control progression through the cell cycle in yeasts 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  42. 1. Enzymes of eukaryotic DNA replication aren’t as well characterized as their prokaryotic counterparts. The replication process is similar in both groups—DNA denatures, replication is semiconservative and semidiscontinuous and primers are required. 2. Five DNA polymerases are known in mammalian cells: a. α (alpha) is nuclear, uses RNA primers, is involved in nuclear DNA replication and has not been shown to proofread. b. β (beta) is nuclear, serves in DNA repair and does not show proofreading activity. c. δ (delta) is nuclear, uses RNA primers, is involved in nuclear DNA replication and is capable of proofreading. d. γ (gamma) is mitochondrial, replicating the mitochondrial DNA using RNA primers and proofreading. e. ε (epsilon) is nuclear, has proofreading activity and may be used for DNA repair. Eukaryotic Replication Enzymes 台大農藝系 遺傳學 601 20000

  43. 1. Eukaryotic chromosomes generally contain much more DNA than those of prokaryotes, and their replication forks move much more slowly. If they were like typical prokaryotes, with only one origin of replication per chromosome, DNA replication would take a very long time. 2. Instead, eukaryotic chromosomes contain multiple origins, at which DNA denatures and replication then proceeds bidirectionally until an adjacent replication fork is encountered. The DNA replicated from a single origin is called a replicon, or replication unit (Figure 3.16). Replicons 台大農藝系 遺傳學 601 20000

  44. Fig. 3.16 Replicating DNA of Drosophila melanogaster 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  45. 3. Replicon size is smaller in eukaryotes than prokaryotes, and each chromosome contains many replicons. Number and size of replicons may vary with cell type. 4. Not all origins within a genome initiate DNA synthesis simultaneously. Cell-specific patterns of origin activation are observed, so that chromosomal regions are replicated in a predictable order in each cell cycle. 台大農藝系 遺傳學 601 20000

  46. Fig. 3.17 Temporal ordering of DNA replication initiation events in replication units of eukaryotic chromosomes 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  47. 1. Eukaryotic origins are generally not well characterized; those of the yeast Saccharomyces cerevisiae are among the best understood. 2. Chromosomal DNA fragments that are able to replicate autonomously when introduced into yeast as extracellular, circular DNA are known as ARSs (autonomously replicating sequences). 3. Some ARSs appear to be yeast origins of replication, but others probably are not. 4. Four sequence elements have been identified in ARSs. The A element is essential to the ARS, and combines with B1 and/or B2 elements to form the core of the ARS to which the multiprotein origin recognition complex (ORC) binds. The B3 element binds a protein called Abf1 (ARS-binding factor) to enhance replication. Origins of Replication 台大農藝系 遺傳學 601 20000

  48. 1. When the ends of chromosomes are replicated and the primers are removed from the 5’ ends, there is no adjacent DNA strand to serve as a primer, and so a single-stranded region is left at the 5’ end of the new strand. If the gap is not addressed, chromosomes would become shorter with each round of replication (Figure 3.18). Replicating the Ends of Chromosomes 台大農藝系 遺傳學 601 20000

  49. Fig. 3.18 The problem of replicating completely a linear chromosome in eukaryotes 台大農藝系 遺傳學 601 20000 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

  50. 2. Most eukaryotic chromosomes have short, species-specific sequences tandemly repeated at their telomeres. Blackburn and Greider have shown that chromosome lengths are maintained by telomerase, which adds telomere repeats without using the cell’s regular replication machinery. 3. In the ciliate Tetrahymena, the telomere repeat sequence is 5’-TTGGGG-3’. Telomerase, an enzyme containing both protein and RNA, binds to the terminal telomere repeat when it is single-stranded, synthesizing a 3-nt sequence, TTG. The 3’ end of the telomerase RNA contains the sequence AAC, which binds the TTG positioning telomerase to complete its synthesis of the TTGGGG telomere repeat. Additional rounds of telomerase activity lengthen the chromosome by adding telomere repeats. 4. After telomerase adds telomere sequences, chromosomal replication proceeds in the usual way. Any shortening of the chromosome ends is compensated by the addition of the telomere repeats. 5. Telomere length may vary, but organisms and cell types have characteristic telomere lengths. Mutants affecting telomere length have been identified, and data indicate that telomere length is genetically controlled. Shortening of telomeres eventually leads to cell death, and this may be a factor in the regulation of normal cell death. 台大農藝系 遺傳學 601 20000

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