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Chapter 11 Study Powerpoints Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
INTRODUCTION <ul><li>DNA replication  is the process by which the genetic material is copied </li></ul><ul><ul><li>The ori...
11.1  STRUCTURAL OVERVIEW  OF DNA REPLICATION <ul><li>DNA replication relies on the complementarity of DNA strands </li></...
T A G C A G A T T A T G G A A C C C T T G C G T A T A C G A T T A C G T A T C G C C G A T C G C A C G G C Incoming nucleot...
Experiment 11A:  Which Model of DNA Replication is Correct? <ul><li>In the late 1950s, three different mechanisms were pro...
Figure 11.2 11-6 (a) Conservative model First round of replication Second round of replication Original double helix (b) S...
<ul><li>In 1958, Matthew Meselson and Franklin Stahl devised a method to investigate these models </li></ul><ul><ul><li>Th...
The Hypothesis <ul><ul><li>Based on Watson’s and Crick’s ideas, the hypothesis was that DNA replication is semiconservativ...
11-9 Figure 11.3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Experimental...
Light Half-heavy Heavy Generations After  14 N Addition 4.1 3.0 2.5 1.9 1.5 1.1 1.0 0.7 0.3 Copyright © The McGraw-Hill Co...
11.2  BACTERIAL  DNA REPLICATION <ul><li>Figure 11.4 presents an overview of the process of bacterial chromosomal replicat...
0.25 μ m (b)  Autoradiograph of an E. coli chromosome in the act of replication (a)  Bacterial chromosome replication Repl...
Initiation of Replication Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><...
11-15 Figure 11.5 E. coli chromosome oriC G G G G G G G A G A G A A A A A A G A A A A T T T T A T T T T T A A T T T T T C ...
AT-rich region DnaA boxes DNA helicase (DnaB protein) binds to the origin. DnaC protein (not shown) assists this process. ...
11-17 Figure 11.6 <ul><ul><li>Composed of six subunits </li></ul></ul><ul><ul><li>Travels along the DNA in the 5’ to 3’ di...
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>DNA helicase separates ...
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-19 Figure 11.7 5′ 3′ 5′ 5′ 3...
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-20
DNA Polymerases Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>DNA pol...
DNA Polymerases Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>DNA pol...
11-23
(a) Schematic side view of DNA polymerase III 3′ 3′ exonuclease site 3′ 5′ 5′ Fingers Thumb DNA polymerase catalytic site ...
11-25 Problem is overcome by making RNA primers using primase DNA polymerases cannot initiate DNA synthesis on a bare temp...
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>The two new daughter st...
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>DNA pol I  removes the ...
11-28 Origin of replication Replication forks Direction of replication fork First Okazaki fragment First and second Okazak...
5′ 3′ 5′ 3′ Origin of replication Replication fork Replication fork Leading strand Lagging strand Leading strand Lagging s...
The Reaction of DNA Polymerase Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display ...
11-31 Figure 11.12 New DNA strand Original DNA strand Copyright © The McGraw-Hill Companies, Inc. Permission required for ...
DNA Polymerase III is a Processive Enzyme Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction ...
DNA Polymerase III is a  Processive Enzyme Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction...
Termination of Replication Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul>...
Fork Fork Fork Fork ter (T2) oriC oriC (T1) Tus Tus Copyright © The McGraw-Hill Companies, Inc. Permission required for re...
Termination of Replication Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul>...
11-37 Figure 11.14 Catenanes Catalyzed by  DNA topoisomerase Replication Decatenation via topoisomerase  Copyright © The M...
DNA Replication Complexes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><...
11-39 Figure 11.15
DNA Replication Complexes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><...
DNA Replication Complexes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><...
Proofreading Mechanisms Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li...
Proofreading Mechanisms Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li...
Proofreading Mechanisms Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li...
11-45 Site where nucleotides are removed from the 3’ end C T 3′ 3′ 5′ 5′ Mismatch causes DNA polymerase to pause, leaving ...
11.3  EUKARYOTIC  DNA REPLICATION <ul><li>Eukaryotic DNA replication is not as well understood as bacterial replication </...
Multiple Origins of Replication Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display...
11-65 Figure 11.22 Chromosome Sister chromatids Before S phase During S phase End of S phase Origin Origin Origin Origin O...
Multiple Origins of Replication Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display...
Multiple Origins of Replication Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display...
Eukaryotes Contain Several Different DNA Polymerases Copyright ©The McGraw-Hill Companies, Inc. Permission required for re...
11-69 *The designations are those of mammalian enzymes. † Many DNA polymerases have dual functions. For example, DNA polym...
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>DNA pol    is the only...
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>DNA polymerases also pl...
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>Flap Endonuclease Remov...
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-73 Figure 11.23 3′ 5′ DNA po...
Telomeres and DNA Replication Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <...
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>Telomeric sequences con...
11-76
<ul><li>DNA polymerases possess two unusual features </li></ul><ul><ul><li>1.  They synthesize DNA only in the 5’ to 3’ di...
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>Therefore if this probl...
11-79 Figure 11.26 Step 1 = Binding Step 3 = Translocation The binding-polymerization-translocation cycle can occurs many ...
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  1. 1. Chapter 11 Study Powerpoints Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
  2. 2. INTRODUCTION <ul><li>DNA replication is the process by which the genetic material is copied </li></ul><ul><ul><li>The original DNA strands are used as templates for the synthesis of new strands </li></ul></ul><ul><li>It occurs very quickly, very accurately, and at the appropriate time in the life of the cell </li></ul><ul><ul><li>This chapter examines how! </li></ul></ul>11-2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
  3. 3. 11.1 STRUCTURAL OVERVIEW OF DNA REPLICATION <ul><li>DNA replication relies on the complementarity of DNA strands </li></ul><ul><ul><li>The AT/GC rule or Chargaff’s rule </li></ul></ul><ul><li>The process can be summarized as follows: </li></ul><ul><ul><li>The two complementary DNA strands come apart </li></ul></ul><ul><ul><li>Each serves as a template strand for the synthesis of new complementary DNA strands </li></ul></ul><ul><ul><li>The two newly-made DNA strands = daughter strands </li></ul></ul><ul><ul><li>The two original DNA strands = parental strands </li></ul></ul>Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-3
  4. 4. T A G C A G A T T A T G G A A C C C T T G C G T A T A C G A T T A C G T A T C G C C G A T C G C A C G G C Incoming nucleotides Original (template) strand Original (template) strand Newly synthesized daughter strand Replication fork (a) The mechanism of DNA replication (b) The products of replication Leading strand Lagging strand 5′ 3′ 3′ 5′ A T A T T A T A T A C G C G G C G C G C G C C G A T 5′ 3′ 5′ 3′ 3′ 5′ A T A T T A T A T A C G C G G C G C G C G C C G A T 3′ 3′ 3′ 5′ A T A T T A T A T A C G C G G C G C G C G C C G A T Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5′ 3′ A A T C Figure 11.1 11-4 Identical base sequences A pairs with T and G pairs with C during synthesis of a new strand
  5. 5. Experiment 11A: Which Model of DNA Replication is Correct? <ul><li>In the late 1950s, three different mechanisms were proposed for the replication of DNA </li></ul><ul><ul><li>Conservative model </li></ul></ul><ul><ul><ul><li>Both parental strands stay together after DNA replication </li></ul></ul></ul><ul><ul><li>Semiconservative model </li></ul></ul><ul><ul><ul><li>The double-stranded DNA contains one parental and one daughter strand following replication </li></ul></ul></ul><ul><ul><li>Dispersive model </li></ul></ul><ul><ul><ul><li>Parental and daughter DNA are interspersed in both strands following replication </li></ul></ul></ul>Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-5
  6. 6. Figure 11.2 11-6 (a) Conservative model First round of replication Second round of replication Original double helix (b) Semiconservative model (c) Dispersive model Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
  7. 7. <ul><li>In 1958, Matthew Meselson and Franklin Stahl devised a method to investigate these models </li></ul><ul><ul><li>They found a way to experimentally distinguish between daughter and parental strands </li></ul></ul><ul><li>Their experiment can be summarized as follows: </li></ul><ul><ul><li>Grow E. coli in the presence of 15 N (a heavy isotope of Nitrogen) for many generations </li></ul></ul><ul><ul><ul><li>The population of cells had heavy-labeled DNA </li></ul></ul></ul><ul><ul><li>Switch E. coli to medium containing only 14 N (a light isotope of Nitrogen) </li></ul></ul><ul><ul><li>Collect sample of cells after various times </li></ul></ul><ul><ul><li>Analyze the density of the DNA by centrifugation using a CsCl gradient </li></ul></ul>Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-7
  8. 8. The Hypothesis <ul><ul><li>Based on Watson’s and Crick’s ideas, the hypothesis was that DNA replication is semiconservative. </li></ul></ul>Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Testing the Hypothesis <ul><ul><li>Refer to Figure 11.3 </li></ul></ul>11-8
  9. 9. 11-9 Figure 11.3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Experimental level Conceptual level 2. Incubate the cells for various lengths of time. Note: The 15 N-labeled DNA is shown in purple and the 14 N-labeled DNA is shown in blue. 3. Lyse the cells by the addition of lysozyme and detergent, which disrupt the bacterial cell wall and cell membrane, respectively. 4. Load a sample of the lysate onto a CsCl gradient. (Note: The average density of DNA is around 1.7 g/cm 3 , which is well isolated from other cellular macromolecules.) 5. Centrifuge the gradients until the DNA molecules reach their equilibrium densities. 6. DNA within the gradient can be observed under a UV light. DNA Cell wall Cell membrane Light DNA Half-heavy DNA Heavy DNA UV light (Result shown here is after 2 generations.) CsCl gradient Lysate Lyse cells 37°C 14 N solution Suspension of bacterial cells labeled with 15 N Up to 4 generations Density centrifugation Generation 0 1 Add 14 N 2 1. Add an excess of 14 N-containing compounds to the bacterial cells so all of the newly made DNA will contain 14 N.
  10. 10. Light Half-heavy Heavy Generations After 14 N Addition 4.1 3.0 2.5 1.9 1.5 1.1 1.0 0.7 0.3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. *Data from: Meselson, M. and Stahl, F.W. (1958) The Replication of DNA in Escherichia coli. Proc. Natl. Acad. Sci. USA 44: 671−682 Interpreting the Data Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-11 After one generation, DNA is “half-heavy” This is consistent with both semi-conservative and dispersive models After ~two generations, DNA is of two types: “ light ” and “ half-heavy ” This is consistent with only the semi-conservative model
  11. 11. 11.2 BACTERIAL DNA REPLICATION <ul><li>Figure 11.4 presents an overview of the process of bacterial chromosomal replication </li></ul><ul><ul><li>DNA synthesis begins at a site termed the origin of replication </li></ul></ul><ul><ul><ul><li>Each bacterial chromosome has only one origin of replication </li></ul></ul></ul><ul><ul><li>Synthesis of DNA proceeds bidirectionally around the bacterial chromosome </li></ul></ul><ul><ul><li>The two replication forks eventually meet at the opposite side of the bacterial chromosome </li></ul></ul><ul><ul><ul><li>This ends replication </li></ul></ul></ul>Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-12
  12. 12. 0.25 μ m (b) Autoradiograph of an E. coli chromosome in the act of replication (a) Bacterial chromosome replication Replication forks Origin of replication Replication fork Site where replication ends Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. From Cold Spring Harbor Symposia of Quantitative Biology, 28, p. 43 (1963). Copyright holder is Cold Spring Habour Laboratory Press. Replication fork 11-13 Figure 11.4
  13. 13. Initiation of Replication Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>The origin of replication in E. coli is termed oriC </li></ul><ul><ul><li>ori gin of C hromosomal replication </li></ul></ul><ul><li>Three types of DNA sequences in oriC are functionally important </li></ul><ul><ul><li>AT-rich region </li></ul></ul><ul><ul><li>DnaA boxes </li></ul></ul><ul><ul><li>GATC methylation sites </li></ul></ul><ul><ul><li>Refer to Figure 11.5 </li></ul></ul>11-14
  14. 14. 11-15 Figure 11.5 E. coli chromosome oriC G G G G G G G A G A G A A A A A A G A A A A T T T T A T T T T T A A T T T T T C T T C A T T C T T C C C 1 C C C C C C T C T C T T T T T T C T T T T A A A T A A A A A T T A A A A A G A A G T A A G A A G G T A G T C C T T A A C A A G G A T A G C C A G T T C C T T T C G DnaA box DnaA box DnaA box DnaA box DnaA box T T G G A T C A T C G C T G G A G G A T C A G G A A T T G T T C C T A T C G G T C A A G G A A G C A A C C T A G T A G C G A C C T C C A T C T A C A T G A A T C C T G G G A A G C A A A A T T G G A A T C T G A A A A C T A T G T G T A A G C C C C G G T T T A C A G C T G G C T T T A T G A A T G A T C G G A G T T A C G G A A A A A A C G A A G G G G C C A A A T G T C G A C C G T A T A C T T A C T A G C C T C A A T G C C T T T T T T G C T T A G C A T A C T G A C G T T C T G T G A G G G T C T A C T C C T G G T T C A T A A C T C T C A A A T C G T A T G A C T A G C A A G A A C C T C C C A G A T G A G G A C C A A G T A T T G A G A G T T T G A T G T A C C A G T A C A G C A T C A G G C A C T A C A T G G T C A T G T A C G T A G T C C G T A G A A T G T A C T T A G G A C C C T T C G T T T T A A C C T T A G A C T T T T G A T A C A C A T C AT-rich region 5′ – – 50 51 100 101 150 201 251 275 250 151 200 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3′
  15. 15. AT-rich region DnaA boxes DNA helicase (DnaB protein) binds to the origin. DnaC protein (not shown) assists this process. DnaA protein Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5′ 3′ AT- rich region 3′ 5′ 5′ 3′ 3′ 5′ 11-16 Figure 11.6 <ul><li>DNA replication is initiated by the binding of DnaA proteins to the DnaA box sequences </li></ul><ul><ul><li>This binding stimulates the cooperative binding of additional ATP-bound DnaA proteins to form a large complex </li></ul></ul><ul><li>Other proteins such as HU and IHF also bind. </li></ul><ul><ul><li>This causes the DNA to wrap around the DnaA proteins causing the separation of the AT-rich region </li></ul></ul>
  16. 16. 11-17 Figure 11.6 <ul><ul><li>Composed of six subunits </li></ul></ul><ul><ul><li>Travels along the DNA in the 5’ to 3’ direction </li></ul></ul><ul><ul><li>Uses energy from ATP </li></ul></ul>Helicase DNA helicase separates the DNA in both directions, creating 2 replication forks. Fork Fork 5′ 3′ 5′ 3′ 3′ 5′ 3′ 5′ <ul><ul><li>Bidirectional replication </li></ul></ul><ul><ul><li>is initiated </li></ul></ul>
  17. 17. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>DNA helicase separates the two DNA strands by breaking the hydrogen bonds between them </li></ul><ul><li>This generates positive supercoiling ahead of each replication fork </li></ul><ul><ul><li>DNA gyrase travels ahead of the helicase and alleviates these supercoils </li></ul></ul><ul><li>Single-strand binding proteins bind to the separated DNA strands to keep them apart </li></ul><ul><li>Then short (10 to 12 nucleotides) RNA primers are synthesized by primase </li></ul><ul><ul><li>These short RNA strands start, or prime, DNA synthesis </li></ul></ul><ul><ul><ul><li>The leading strand has a single primer, the lagging strand needs multiple primers </li></ul></ul></ul><ul><ul><ul><li>They are eventually removed and replaced with DNA </li></ul></ul></ul>11-18
  18. 18. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-19 Figure 11.7 5′ 3′ 5′ 5′ 3′ 3′ DNA polymerase III Origin Leading strand Lagging strand Linked Okazaki fragments Direction of fork movement Functions of key proteins involved with DNA replication DNA polymerase III RNA primer Okazaki fragment DNA ligase RNA primer Single-strand binding protein DNA helicase Topoisomerase Parental DNA Primase Replication fork Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. • DNA helicase breaks the hydrogen bonds between the DNA strands. • Topoisomerase alleviates positive supercoiling. • Single-strand binding proteins keep the parental strands apart. • Primase synthesizes an RNA primer. • DNA polymerase III synthesizes a daughter strand of DNA. • DNA polymerase I excises the RNA primers and fills in with DNA (not shown). • DNA ligase covalently links the Okazaki fragments together.
  19. 19. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-20
  20. 20. DNA Polymerases Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>DNA polymerases are the enzymes that catalyze the attachment of nucleotides to synthesize a new DNA strand </li></ul><ul><li>In E. coli there are five proteins with polymerase activity </li></ul><ul><ul><li>DNA pol I, II, III, IV and V </li></ul></ul><ul><ul><li>DNA pol I and III </li></ul></ul><ul><ul><ul><li>Normal replication </li></ul></ul></ul><ul><ul><li>DNA pol II, IV and V </li></ul></ul><ul><ul><ul><li>DNA repair and replication of damaged DNA </li></ul></ul></ul>11-21
  21. 21. DNA Polymerases Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>DNA pol I </li></ul><ul><ul><li>Composed of a single polypeptide </li></ul></ul><ul><ul><li>Removes the RNA primers and replaces them with DNA </li></ul></ul><ul><li>DNA pol III </li></ul><ul><ul><li>Responsible for most of the DNA replication </li></ul></ul><ul><ul><li>Composed of 10 different subunits (Table 11.2) </li></ul></ul><ul><ul><ul><li>The  subunit catalyzes bond formation between adjacent nucleotides (DNA synthesis) </li></ul></ul></ul><ul><ul><ul><li>The other 9 fulfill other functions </li></ul></ul></ul><ul><ul><li>The complex of all 10 subunits is referred to as DNA polymerase holoenzyme </li></ul></ul>11-22
  22. 22. 11-23
  23. 23. (a) Schematic side view of DNA polymerase III 3′ 3′ exonuclease site 3′ 5′ 5′ Fingers Thumb DNA polymerase catalytic site Template strand Palm Incoming deoxyribonucleoside triphosphates (dNTPs) Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>Bacterial DNA polymerases may vary in their subunit composition </li></ul><ul><ul><li>However, they all have the same type of catalytic subunit </li></ul></ul>11-24 Figure 11.8 Structure resembles a human right hand Template DNA is threaded through the palm Thumb and fingers wrapped around the DNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
  24. 24. 11-25 Problem is overcome by making RNA primers using primase DNA polymerases cannot initiate DNA synthesis on a bare template strand DNA polymerases can attach nucleotides only in the 5’ to 3’ direction Problem is overcome by synthesizing the new strands both toward, and away from, the replication fork (b) (a) 3′ 5′ 5′ 3′ 3′ 5′ 5′ 3′ Cannot link nucleotides in this direction Able to covalently link together Can link nucleotides in this direction Unable to covalently link the 2 individual nucleotides together Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Primer Unusual features of DNA polymerase function Figure 11.9
  25. 25. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>The two new daughter strands are synthesized in different ways </li></ul><ul><ul><li>Leading strand </li></ul></ul><ul><ul><ul><li>One RNA primer is made at the origin </li></ul></ul></ul><ul><ul><ul><li>DNA pol III attaches nucleotides in a 5’ to 3’ direction as it slides toward the opening of the replication fork </li></ul></ul></ul><ul><ul><li>Lagging strand </li></ul></ul><ul><ul><ul><li>Synthesis is also in the 5’ to 3’ direction </li></ul></ul></ul><ul><ul><ul><ul><li>However it occurs away from the replication fork </li></ul></ul></ul></ul><ul><ul><ul><li>Many RNA primers are required </li></ul></ul></ul><ul><ul><ul><li>DNA pol III uses the RNA primers to synthesize small DNA fragments (1000 to 2000 nucleotides each) </li></ul></ul></ul><ul><ul><ul><ul><li>These are termed Okazaki fragments after their discoverers </li></ul></ul></ul></ul>11-26
  26. 26. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>DNA pol I removes the RNA primers and fills the resulting gap with DNA </li></ul><ul><ul><li>Uses its 5’ to 3’ exonuclease activity to digest the RNA </li></ul></ul><ul><ul><li>Uses its 5’ to 3’ polymerase activity to replace it with DNA </li></ul></ul><ul><li>After the gap is filled a covalent bond is still missing </li></ul><ul><li>DNA ligase catalyzes the formation of a covalent phosphoester bond </li></ul><ul><ul><li>Thereby connecting the DNA fragments </li></ul></ul>11-27
  27. 27. 11-28 Origin of replication Replication forks Direction of replication fork First Okazaki fragment First and second Okazaki fragments have been connected to each other. First Okazaki fragment of the lagging strand Second Okazaki fragment Third Okazaki fragment Primer Primer The leading strand elongates, and a second Okazaki fragment is made. The leading strand continues to elongate. A third Okazaki fragment is made, and the first and second are connected together. Primers are needed to initiate DNA synthesis. The synthesis of the leading strand occurs in the same direction as the movement of the replication fork. The first Okazaki fragment of the lagging strand is made in the opposite direction. 5′ 5′ 5′ 5′ 3′ 5′ 3′ 3′ 5′ 3′ 3′ 3′ 5′ 5′ 5′ 5′ 3′ 3′ 3′ 3′ 5′ 5′ 3′ 3′ Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Leading strand DNA strands separate at origin, creating 2 replication forks. Figure 11.10
  28. 28. 5′ 3′ 5′ 3′ Origin of replication Replication fork Replication fork Leading strand Lagging strand Leading strand Lagging strand Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 11-29 Figure 11.11 The synthesis of leading and lagging strands from a single origin of replication
  29. 29. The Reaction of DNA Polymerase Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>DNA polymerases catalyzes the formation of a covalent (phosphoester) bond between the </li></ul><ul><ul><li>Innermost phosphate group of the incoming deoxyribonucleoside triphosphate </li></ul></ul><ul><ul><ul><li>and </li></ul></ul></ul><ul><ul><li>3’-OH of the sugar of the previous deoxynucleotide </li></ul></ul><ul><li>In the process, the last two phosphates of the incoming nucleotide are released </li></ul><ul><ul><li>In the form of pyrophosphate (PP i ) </li></ul></ul><ul><ul><li>Refer to figure 11.12 </li></ul></ul>11-30
  30. 30. 11-31 Figure 11.12 New DNA strand Original DNA strand Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. O O O O O O P CH 2 O – Incoming nucleotide (a deoxyribonucleoside triphosphate) Pyrophosphate (PPi) New ester bond 5′ 5′ end O O O P CH 2 O – O O O CH 2 O – O O O O – P O – 5′ O O O O O P CH 2 5′ 5′ end 3′ end O O P CH 2 O – O O O O P CH 2 O – O O P O – O P O – O – O – + OH 3′ OH 3′ OH 3′ P P O Cytosine Guanine Guanine Cytosine Thymine Adenine Cytosine Guanine Guanine Cytosine Thymine Adenine O O O O O P H 2 C H 2 C O – 3′ O O O P O O H 2 C O – O O P O – 5′ 5′ end O O O O O P H 2 C H 2 C O – 3′ O O O P O O H 2 C O – O O P O – 5′ 3′ end 5′ end O O – O – 3′ end Innermost phosphate
  31. 31. DNA Polymerase III is a Processive Enzyme Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>DNA polymerase III remains attached to the template as it is synthesizing the daughter strand </li></ul><ul><li>This processive feature is due to several different subunits in the DNA pol III holoenzyme </li></ul><ul><ul><li> subunit forms a dimer in the shape of a ring around template DNA </li></ul></ul><ul><ul><ul><li>It is termed the clamp protein </li></ul></ul></ul><ul><ul><ul><li>Once bound, the  subunits can freely slide along dsDNA </li></ul></ul></ul><ul><ul><ul><li>Promotes association of holoenzyme with DNA </li></ul></ul></ul><ul><ul><li> complex catalyzes  dimer clamping to the DNA </li></ul></ul><ul><ul><ul><li>It is termed the clamp-loader complex </li></ul></ul></ul><ul><ul><ul><li>Includes  ’,  and  subunits </li></ul></ul></ul>11-32
  32. 32. DNA Polymerase III is a Processive Enzyme Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>The effect of processivity is quite remarkable </li></ul><ul><ul><li>In the absence of the  subunit </li></ul></ul><ul><ul><ul><li>DNA pol III falls off the DNA template after about 10 nucleotides have been polymerized </li></ul></ul></ul><ul><ul><ul><li>Its rate is ~ 20 nucleotides per second </li></ul></ul></ul><ul><ul><li>In the presence of the  subunit </li></ul></ul><ul><ul><ul><li>DNA pol III stays on the DNA template long enough to polymerize up to 500,000 nucleotides </li></ul></ul></ul><ul><ul><ul><li>Its rate is ~ 750 nucleotides per second </li></ul></ul></ul>11-33
  33. 33. Termination of Replication Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>On the opposite side of the chromosome to oriC is a pair of termination sequences called ter sequences </li></ul><ul><ul><li>These are designated T1 and T2 </li></ul></ul><ul><ul><ul><li>T1 stops counterclockwise forks, T2 stops clockwise forks </li></ul></ul></ul><ul><li>The protein tus ( t ermination u tilization s ubstance) binds to the ter sequences </li></ul><ul><ul><li>tus bound to the ter sequences stops the movement of the replication forks </li></ul></ul><ul><li>Refer to Figure 11.13 </li></ul>11-34
  34. 34. Fork Fork Fork Fork ter (T2) oriC oriC (T1) Tus Tus Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. ter 11-35 Figure 11.13 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Prevents advancement of fork moving right-to-left (counterclockwise fork) Prevents advancement of fork moving left-to-right (clockwise fork)
  35. 35. Termination of Replication Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>DNA replication ends when oppositely advancing forks meet (usually at T1 or T2) </li></ul><ul><li>Finally DNA ligase covalently links the two daughter strands </li></ul><ul><li>DNA replication often results in two intertwined molecules </li></ul><ul><ul><li>Intertwined circular molecules are termed catenanes </li></ul></ul><ul><ul><li>These are separated by the action of topoisomerase </li></ul></ul>11-36
  36. 36. 11-37 Figure 11.14 Catenanes Catalyzed by DNA topoisomerase Replication Decatenation via topoisomerase Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
  37. 37. DNA Replication Complexes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>DNA helicase and primase are physically bound to each other to form a complex called the primosome </li></ul><ul><ul><li>This complex leads the way at the replication fork </li></ul></ul><ul><li>The primosome is physically associated with two DNA polymerase holoenzymes to form the replisome </li></ul><ul><li>Figure 11.15 provides a three-dimensional view of DNA replication </li></ul>11-38
  38. 38. 11-39 Figure 11.15
  39. 39. DNA Replication Complexes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>Two DNA pol III proteins act in concert to replicate both the leading and lagging strands </li></ul><ul><ul><li>The two proteins form a dimeric DNA polymerase that moves as a unit toward the replication fork </li></ul></ul><ul><li>DNA polymerases can only synthesize DNA in the 5’ to 3’ direction </li></ul><ul><ul><li>Synthesis of the leading strand is continuous </li></ul></ul><ul><ul><li>Synthesis of the lagging strand is discontinuous </li></ul></ul>11-40
  40. 40. DNA Replication Complexes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>Lagging strand synthesis is summarized as follows: </li></ul><ul><ul><li>The lagging strand is looped </li></ul></ul><ul><ul><ul><li>This allows the attached DNA polymerase to synthesize the Okazaki fragments in the 5’ to 3’ direction yet move toward the fork </li></ul></ul></ul><ul><li>Upon completion of an Okazaki fragment, DNA polymerase releases the lagging template strand </li></ul><ul><ul><ul><li>The clamp loader complex then reloads the polymerase at the next RNA primer </li></ul></ul></ul><ul><ul><ul><li>Another loop is then formed </li></ul></ul></ul><ul><ul><li>This process is repeated over and over again </li></ul></ul>11-41
  41. 41. Proofreading Mechanisms Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>DNA replication exhibits a high degree of fidelity </li></ul><ul><ul><li>Mistakes during the process are extremely rare </li></ul></ul><ul><ul><ul><li>DNA pol III makes only one mistake per 10 8 bases made </li></ul></ul></ul><ul><li>There are three reasons why fidelity is high </li></ul><ul><ul><li>1. Instability of mismatched pairs </li></ul></ul><ul><ul><li>2. Configuration of the DNA polymerase active site </li></ul></ul><ul><ul><li>3. Proofreading function of DNA polymerase </li></ul></ul>11-42
  42. 42. Proofreading Mechanisms Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>1. Instability of mismatched pairs </li></ul><ul><ul><li>Complementary base pairs have much higher stability than mismatched pairs </li></ul></ul><ul><ul><li>Stability of base pairs only accounts for part of the fidelity </li></ul></ul><ul><ul><ul><li>Error rate for mismatched base pairs is 1 per 1,000 nucleotides </li></ul></ul></ul><ul><li>2. Configuration of the DNA polymerase active site </li></ul><ul><ul><li>DNA polymerase is unlikely to catalyze bond formation between mismatched pairs </li></ul></ul><ul><ul><li>This induced-fit phenomenon decreases the error rate to a range of 1 in 100,000 to 1 million </li></ul></ul>11-43
  43. 43. Proofreading Mechanisms Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>3. Proofreading function of DNA polymerase </li></ul><ul><ul><li>DNA polymerases can identify a mismatched nucleotide and remove it from the daughter strand </li></ul></ul><ul><ul><li>The enzyme uses a 3’ to 5’ exonuclease activity to digest the newly made strand until the mismatched nucleotide is removed </li></ul></ul><ul><ul><li>DNA synthesis then resumes in the 5’ to 3’ direction </li></ul></ul><ul><ul><ul><li>Refer to figure 11.16 </li></ul></ul></ul>11-44
  44. 44. 11-45 Site where nucleotides are removed from the 3’ end C T 3′ 3′ 5′ 5′ Mismatch causes DNA polymerase to pause, leaving mismatched nucleotide near the 3′ end. Template strand The 3′ end enters the exonuclease site. 3′ 5′ 5′ At the 3′ exonuclease site, the strand is digested in the 3′ to 5′ direction until the incorrect nucleotide is removed. 3′ 5′ 5′ Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Base pair mismatch near the 3′ end 3′ 3′ Incorrect nucleotide removed exonuclease site A schematic drawing of proofreading Figure 11.16
  45. 45. 11.3 EUKARYOTIC DNA REPLICATION <ul><li>Eukaryotic DNA replication is not as well understood as bacterial replication </li></ul><ul><ul><li>The two processes do have extensive similarities </li></ul></ul><ul><ul><ul><li>The types of bacterial enzymes described in Table 11.1 have also been found in eukaryotes </li></ul></ul></ul><ul><ul><li>Nevertheless, DNA replication in eukaryotes is more complex </li></ul></ul><ul><ul><ul><li>Large linear chromosomes </li></ul></ul></ul><ul><ul><ul><li>Chromatin is tightly packed within nucleosomes </li></ul></ul></ul><ul><ul><ul><li>More complicated cell cycle regulation </li></ul></ul></ul>Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-62
  46. 46. Multiple Origins of Replication Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>Eukaryotes have long linear chromosomes </li></ul><ul><ul><li>They therefore require multiple origins of replication </li></ul></ul><ul><ul><ul><li>To ensure that the DNA can be replicated in a reasonable amount of time </li></ul></ul></ul><ul><li>In 1968, Huberman and Riggs provided evidence for multiple origins of replication </li></ul><ul><ul><li>Refer to Figure 11.21 </li></ul></ul><ul><li>DNA replication proceeds bidirectionally from many origins of replication </li></ul><ul><ul><li>Refer to Figure 11.22 </li></ul></ul>11-63
  47. 47. 11-65 Figure 11.22 Chromosome Sister chromatids Before S phase During S phase End of S phase Origin Origin Origin Origin Origin Centromere Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
  48. 48. Multiple Origins of Replication Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>The origins of replication found in eukaryotes have some similarities to those of bacteria </li></ul><ul><ul><li>Origins of replication in Saccharomyces cerevisiae are termed ARS elements ( A utonomously R eplicating S equence) </li></ul></ul><ul><ul><ul><li>They are about 50 bp in length </li></ul></ul></ul><ul><ul><ul><li>They have a high percentage of A and T </li></ul></ul></ul><ul><ul><ul><li>ARS consensus sequence (ACS) </li></ul></ul></ul><ul><ul><ul><ul><li>ATTTAT(A or G)TTTA </li></ul></ul></ul></ul>11-66
  49. 49. Multiple Origins of Replication Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>Replication begins with assembly of the prereplication complex (preRC) </li></ul><ul><ul><li>Consists of at least 14 different proteins </li></ul></ul><ul><li>An important part of the preRC is the Origin recognition complex (ORC) </li></ul><ul><ul><li>A six-subunit complex that acts as the initiator of eukaryotic DNA replication </li></ul></ul><ul><li>Other preRC proteins include MCM Helicase </li></ul><ul><ul><li>Binding of MCM completes DNA replication licensing </li></ul></ul><ul><ul><ul><li>The origin becomes capable of initiating DNA synthesis </li></ul></ul></ul><ul><ul><li>Binding of at least 22 additional proteins is required to initiate synthesis during S phase </li></ul></ul>11-67
  50. 50. Eukaryotes Contain Several Different DNA Polymerases Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>Mammalian cells contain well over a dozen different DNA polymerases </li></ul><ul><ul><li>Refer to Table 11.4 </li></ul></ul><ul><li>Four: alpha (  ), delta (  ), epsilon (  ) and gamma (  ) have the primary function of replicating DNA </li></ul><ul><ul><li> ,  and    Nuclear DNA </li></ul></ul><ul><ul><li>  Mitochondrial DNA </li></ul></ul>11-68
  51. 51. 11-69 *The designations are those of mammalian enzymes. † Many DNA polymerases have dual functions. For example, DNA polymerases α, δ, and ε are involved in the replication of normal DNA and also play a role in DNA repair. In cells of the immune system, certain genes that encode antibodies (i.e., immunoglobulin genes) undergo a phenomenon known as hypermutation. This increases the variation in the kinds of antibodies the cells can make. Certain polymerases in this list, such as η, may play a role in hypermutation of immunoglobulin genes. DNA polymerase σ may play a role in sister chromatid cohesion, a topic discussed in Chapter 10 .
  52. 52. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>DNA pol  is the only polymerase to associate with primase </li></ul><ul><ul><li>The DNA pol  /primase complex synthesizes a short RNA-DNA hybrid primer </li></ul></ul><ul><ul><ul><li>10 RNA nucleotides followed by 20 to 30 DNA nucleotides </li></ul></ul></ul><ul><ul><li>This hybrid primer is used by DNA pol  or  for the processive elongation of the leading and lagging strands, respectively </li></ul></ul><ul><li>The exchange of DNA pol  for  or  is required for elongation of the leading and lagging strands. </li></ul><ul><ul><li>This is called a polymerase switch </li></ul></ul><ul><ul><ul><li>It occurs only after the RNA-DNA hybrid is made </li></ul></ul></ul>11-70
  53. 53. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>DNA polymerases also play a role in DNA repair </li></ul><ul><ul><li>DNA pol  is not involved in DNA replication </li></ul></ul><ul><ul><li>It plays a role in base-excision repair </li></ul></ul><ul><ul><ul><li>Removal of incorrect bases from damaged DNA </li></ul></ul></ul><ul><li>Recently, more DNA polymerases have been identified </li></ul><ul><ul><li>Lesion-replicating polymerases </li></ul></ul><ul><ul><ul><li>Involved in the replication of damaged DNA </li></ul></ul></ul><ul><ul><ul><li>They can synthesize a complementary strand over the abnormal region </li></ul></ul></ul>11-71
  54. 54. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>Flap Endonuclease Removes RNA Primers </li></ul><ul><ul><li>Polymerase  runs into primer of adjacent Okazaki fragment </li></ul></ul><ul><ul><ul><li>Pushes portion of primer into a short flap </li></ul></ul></ul><ul><ul><ul><li>Flap endonuclease removes the primer </li></ul></ul></ul><ul><ul><ul><li>Long flaps are shortened by Dna2 nuclease/helicase </li></ul></ul></ul><ul><ul><ul><li>Refer to Figure 11.23 </li></ul></ul></ul>11-72
  55. 55. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-73 Figure 11.23 3′ 5′ DNA polymerase δ elongates the left Okazaki fragment and causes a short flap to occur on the right Okazaki fragment. Flap 5′ 3′ 3′ 5′ Process continues until the entire RNA primer is removed. DNA ligase seals the two fragments together. 5′ 3′ Flap endonuclease removes the flap. 5′ 3′ 3′ 5′ DNA polymerase δ continues to elongate and causes a second flap. 5′ 3′ 3′ 5′ Flap endonuclease removes the flap. 5′ 3′ 3′ 5′ 5′ 3′ 3′ 5′ Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
  56. 56. Telomeres and DNA Replication Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>Linear eukaryotic chromosomes have telomeres at both ends </li></ul><ul><li>The term telomere refers to the complex of telomeric DNA sequences and bound proteins </li></ul>11-74
  57. 57. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>Telomeric sequences consist of </li></ul><ul><ul><li>Moderately repetitive tandem arrays </li></ul></ul><ul><ul><li>3’ overhang that is 12-16 nucleotides long </li></ul></ul>11-75 Figure 11.24 <ul><li>Telomeric sequences typically consist of </li></ul><ul><ul><li>Several guanine nucleotides </li></ul></ul><ul><ul><li>Many thymine nucleotides </li></ul></ul><ul><li>Refer to Table 11.5 </li></ul>Telomeric repeat sequences Overhang C G C G C G T A A T A T C G C G C G T A A T A T C G C G C G T A A T A T C G C G C G T A A T A T C G C G C G T A A T A T C G C G C G T A A T A T C G C G C G T A A T T A T G G G A A T A T G G G A T A T T T G G G 5′ 3′ Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
  58. 58. 11-76
  59. 59. <ul><li>DNA polymerases possess two unusual features </li></ul><ul><ul><li>1. They synthesize DNA only in the 5’ to 3’ direction </li></ul></ul><ul><ul><li>2. They cannot initiate DNA synthesis </li></ul></ul><ul><li>These two features pose a problem at the 3’ ends of linear chromosomes-the end of the strand cannot be replicated! </li></ul>11-77 Figure 11.25 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA polymerase cannot link these two nucleotides together without a primer. No place for a primer 3′ 5′
  60. 60. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display <ul><li>Therefore if this problem is not solved </li></ul><ul><ul><li>The linear chromosome becomes progressively shorter with each round of DNA replication </li></ul></ul><ul><li>The cell solves this problem by adding DNA sequences to the ends of telomeres </li></ul><ul><li>This requires a specialized mechanism catalyzed by the enzyme telomerase </li></ul><ul><li>Telomerase contains protein and RNA </li></ul><ul><ul><li>The RNA is complementary to the DNA sequence found in the telomeric repeat </li></ul></ul><ul><ul><ul><li>This allows the telomerase to bind to the 3’ overhang </li></ul></ul></ul><ul><ul><li>The lengthening mechanism is outlined in Figure 11.26 </li></ul></ul>11-78
  61. 61. 11-79 Figure 11.26 Step 1 = Binding Step 3 = Translocation The binding-polymerization-translocation cycle can occurs many times This greatly lengthens one of the strands The end is now copied Step 2 = Polymerization RNA primer is made and other strand is synthesized. Telomerase reverse transcriptase (TERT) activity Telomere Telomerase Eukaryotic chromosome Repeat unit 3′ 3 5′ T T A G G G T T A A A T C C C A A T C C C A A U C C C G G G A G G G T T A T T G G G T T A G G G T T A C C C A A U C C C G G G T T A T T G G G T T A G G G A G G G C C C A A U C C C T T C C C A A T A A A A T C C C U A A C U C C C C C T T A G G G T T A G G G T T A T T G T T A G G G A G G G G G T T A G G G T T A G G G T T A T T G T T A G G G A G G G G G G T T A G G A A T C C C A A T A A T C C C A A T A A T C C C A A T RNA RNA primer Telomerase synthesizes a 6-nucleotide repeat. Telomerase moves 6 nucleotides to the right and begins to make another repeat. The complementary strand is made by primase, DNA polymerase, and ligase. 3′ 5′ 5′ 3′ 3′ Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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