1. Chapter 11 Study Powerpoints Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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

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.

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. 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

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

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′

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. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-20

22. 11-23

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

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. 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

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

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)

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.

38. 11-39 Figure 11.15

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

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.

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 .

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.

58. 11-76

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.