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1 PowerPoint Presentation Materials to accompany
Genetics: Analysis and Principles Robert J. Brooker CHAPTER 11 DNA REPLICATION Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

2 11.1 STRUCTURAL OVERVIEW OF DNA REPLICATION
DNA replication relies on the complementarity of DNA strands The AT/GC rule or Chargaff’s rule The process can be summarized as such The two DNA strands come apart Each serves as a template strand for the synthesis of new strands The two newly-made strands = daughter strands The two original ones = parental strands 11-3 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

3 Identical base sequences
Figure 11.1 11-4

4 Experiment 11A: Which Model of DNA Replication is Correct?
Conservative model Both parental strands stay together after DNA replication Semiconservative model The double-stranded DNA contains one parental and one daughter strand following replication Dispersive model Parental and daughter DNA are interspersed in both strands following replication 11-5 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

5 Figure 11-2 Copyright © 2006 Pearson Prentice Hall, Inc.
Figure 11-2 Results of one round of replication of DNA for each of the three possible modes by which replication could be accomplished. Figure Copyright © 2006 Pearson Prentice Hall, Inc.

6 1958: Matthew Meselson and Franklin Stahl
Experimentally distinguish between daughter and parental strands Grow E. coli in the presence of 15N (a heavy isotope of Nitrogen) for many generations The population of cells had heavy-labeled DNA Switch E. coli to medium containing only 14N (a light isotope of Nitrogen) Collect sample of cells after various times Analyze the density of the DNA by centrifugation using a CsCl gradient 11-7 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

7 Testing the Hypothesis
This experiment aims to determine which of the three models of DNA replication is correct Testing the Hypothesis Refer to Figure 11.3 11-8 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

8 Figure 11.3 11-9

9 DNA Figure 11.3 11-10

10 Figure 11-3 The Meselson–Stahl experiment.

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

12 Interpreting the Data 11-12 After one generation, DNA is “half-heavy”
After ~ two generations, DNA is of two types: “light” and “half-heavy” This is consistent with both semi-conservative and dispersive models This is consistent with only the semi-conservative model 11-12 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

13 11.2 BACTERIAL DNA REPLICATION
Figure 11.4 presents an overview of the process of bacterial chromosome replication DNA synthesis begins at a site termed the origin of replication Each bacterial chromosome has only one Synthesis of DNA proceeds bidirectionally around the bacterial chromosome The replication forks eventually meet at the opposite side of the bacterial chromosome This ends replication 11-13 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

14 Figure 11.4 11-14 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

15 Initiation of Replication
The origin of replication in E. coli is termed oriC origin of Chromosomal replication Three types of DNA sequences in oriC are functionally significant AT-rich region DnaA boxes GATC methylation sites Refer to Figure 11.5 11-15 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

16 Figure 11.5 11-16 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

17 11-17 Figure 11.6 Other proteins such as HU and IHF also bind.
This causes the region to wrap around the DnaA proteins and separates the AT-rich region DNA replication is initiated by the binding of DnaA proteins to the DnaA box sequences This binding stimulates the cooperative binding of an additional 20 to 40 DnaA proteins to form a large complex 11-17

18 Travels along the DNA in the 5’ to 3’ direction
Figure 11.6 Composed of six subunits Travels along the DNA in the 5’ to 3’ direction Uses energy from ATP Bidirectional replication 11-18

19 11-20 Figure 11.7 Keep the parental strands apart
Breaks the hydrogen bonds between the two strands Alleviates supercoiling Synthesizes an RNA primer Figure 11.7 11-20 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

20 DNA Polymerases DNA polymerases are the enzymes that catalyze the attachment of nucleotides to make new DNA In E. coli there are five proteins with polymerase activity DNA pol I and III Normal replication DNA pol II, IV and V DNA repair and replication of damaged DNA 11-21 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

21 DNA polymerase catalyzes DNA synthesis and requires a DNA template and all four dNTPs
DNA polymerases can elongate an existing DNA strand (called a primer) but cannot initiate DNA synthesis.

22 Figure 11-10 Copyright © 2006 Pearson Prentice Hall, Inc.
The enzyme primase synthesizes an RNA primer that provides the free 3'-hydroxyl required by DNA polymerase III Figure The initiation of DNA synthesis. A complementary RNA primer is first synthesized, to which DNA is added. All synthesis is in the to direction. Eventually, the RNA primer is replaced with DNA under the direction of DNA polymerase I. Figure Copyright © 2006 Pearson Prentice Hall, Inc.

23 11-25 Figure 11.9 Unusual features of DNA polymerase function
DNA polymerases cannot initiate DNA synthesis Problem is overcome by the RNA primers synthesized by primase Problem is overcome by synthesizing the 3’ to 5’ strands in small fragments DNA polymerases can attach nucleotides only in the 5’ to 3’ direction Unusual features of DNA polymerase function Figure 11.9 11-25

24 The two new daughter strands are synthesized in different ways
Leading strand One RNA primer is made at the origin DNA pol III attaches nucleotides in a 5’ to 3’ direction as it slides toward the opening of the replication fork Lagging strand Synthesis is also in the 5’ to 3’ direction However it occurs away from the replication fork Many RNA primers are required DNA pol III uses the RNA primers to synthesize small DNA fragments (1000 to 2000 nucleotides each) These are termed Okazaki fragments after their discoverers 11-26 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

25 DNA pol I removes the RNA primers and fills the resulting gap with DNA
It uses its 5’ to 3’ exonuclease activity to digest the RNA and its 5’ to 3’ polymerase activity to replace it with DNA After the gap is filled a covalent bond is still missing DNA ligase catalyzes a phosphodiester bond Thereby connecting the DNA fragments 11-27 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

26 11-28 Figure 11.7 Keep the parental strands apart
Breaks the hydrogen bonds between the two strands Alleviates supercoiling Keep the parental strands apart Synthesizes an RNA primer Synthesizes daughter DNA strands III Covalently links DNA fragments together 11-28 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

27 The Reaction of DNA Polymerase
DNA polymerases catalyzes a phosphodiester bond between the Innermost phosphate group of the incoming deoxynucleoside triphosphate AND 3’-OH of the sugar of the previous deoxynucleotide 11-29 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

28 Figure 11-8 Copyright © 2006 Pearson Prentice Hall, Inc.
Figure 11-8 Demonstration of to synthesis of DNA. Figure Copyright © 2006 Pearson Prentice Hall, Inc.

29 Figure 11.10 Innermost phosphate 11-30

30 Termination of Replication
DNA replication ends when oppositely advancing forks meet (usually at T1 or T2) Finally DNA ligase covalently links all four DNA strands DNA replication often results in two intertwined molecules Intertwined circular molecules are termed catenanes These are separated by the action of topoisomerases 11-35 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

31 Catalyzed by DNA topoisomerases
Figure 11.12 Catenanes Catalyzed by DNA topoisomerases 11-36 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

32 QUICK REVIEW 11-37 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

33 DNA Replication Complexes
Two DNA pol III proteins act in concert to replicate both the leading and lagging strands The two proteins form a dimeric DNA polymerase that moves as a unit toward the replication fork DNA polymerases can only synthesize DNA in the 5’ to 3’ direction So synthesis of the leading strand is continuous And that of the lagging strand is discontinuous 11-40 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

34 DNA Replication Complexes
Lagging strand synthesis is summarized as such: The lagging strand is looped This allows the attached DNA polymerase to synthesize the Okazaki fragments in the normal 5’ to 3’ direction Upon completion of an Okazaki fragment, the enzyme releases the lagging template strand Another loop is then formed This processed is repeated over and over again 11-41 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

35 DNA Replication Complexes
Figure 11.13 DNA Replication Complexes 11-39 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

36 Proofreading Mechanisms
DNA replication exhibits a high degree of fidelity Mistakes during the process are extremely rare DNA pol III makes only one mistake per 108 bases made There are several reasons why fidelity is high 1. Instability of mismatched pairs 2. Configuration of the DNA polymerase active site 3. Proofreading function of DNA polymerase 11-42 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

37 Proofreading Mechanisms
1. Instability of mismatched pairs Complementary base pairs have much higher stability than mismatched pairs This feature only accounts for part of the fidelity It has an error rate of 1 per 1,000 nucleotides 2. Configuration of the DNA polymerase active site DNA polymerase is unlikely to catalyze bond formation between mismatched pairs This induced-fit phenomenon decreases the error rate to a range of 1 in 100,000 to 1 million 11-43 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

38 Proofreading Mechanisms
3. Proofreading function of DNA polymerase DNA polymerases can identify a mismatched nucleotide and remove it from the daughter strand The enzyme uses its 3’ to 5’ exonuclease activity to remove the incorrect nucleotide It then changes direction and resumes DNA synthesis in the 5’ to 3’ direction 11-44 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

39 11-45 Figure 11.14 A schematic drawing of proofreading
Site where DNA backbone is cut A schematic drawing of proofreading Figure 11.14 11-45 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

40 Experiment 11B: DNA Replication Can Be Studied In Vitro
The in vitro study of DNA replication was pioneered by Arthur Kornberg in the 1950s He received a Nobel Prize for his efforts in 1959 Kornberg hypothesized that deoxynucleoside triphosphates are the precursors of DNA synthesis 11-50 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

41 Experiment 11B: DNA Replication Can Be Studied In Vitro
In this experiment, Kornberg mixed the following An extract of proteins from E. coli Template DNA Radiolabeled nucleotides These were incubated for sufficient time to allow the synthesis of new DNA strands Addition of acid will precipitate these DNA strands Centrifugation will separate them from the radioactive nucleotides 11-51 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

42 Testing the Hypothesis
DNA synthesis can occur in vitro if all the necessary components are present Testing the Hypothesis Refer to Figure 11.17 11-52 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

43 Figure 11.17 11-53

44 Figure 11.17 11-54

45 The Data 11-55 Conditions Amount of radiolabeled DNA* Complete system
3,300 Template DNA omitted 0.0 *Calculated in picomoles of 32P-labeled DNA 11-55 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

46 Interpreting the Data 11-56 Conditions Amount of radiolabeled DNA*
E. coli proteins + nonlabeled template DNA + radiolabled nucleotides = Radiolabeled product Conditions Amount of radiolabeled DNA* Complete system 3,300 Template DNA omitted 0.0 *Calculated in picomoles of 32P-labeled DNA Expected result because the template is necessary for replication Taken together, these results indicate that this technique can be used to measure the synthesis of DNA in vitro 11-56 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

47 Isolation of Mutants The isolation of mutants has been crucial in elucidating DNA replication For example, mutants played key roles in the discovery of DNA pol III Various other enzymes involved in DNA synthesis DNA replication is vital for cell division Thus, most mutations that block DNA synthesis are lethal For this reason, researchers must screen for conditional mutants 11-57 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

48 Isolation of Mutants A type of conditional mutant is a temperature-sensitive (ts) mutant In the case of a vital gene A ts mutant can survive at the permissive temperature But it will fail to grow at the nonpermissive one Figure shows a general strategy for the isolation of ts mutants 11-58 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

49 To increase the likelihood of mutations
Figure 11.18 11-59

50 Isolation of Mutants E. coli has many vital genes that are not involved in DNA replication So only a subset of ts mutants would carry mutations affecting the replication process Therefore, researchers in the 1960s had to screen thousands of ts mutants to get to those involved in DNA replication This is sometimes called a “brute force” genetic screen Table 11.3 summarizes some of the genes that were identified using this strategy 11-60 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

51 11-61 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

52 Isolation of Mutants The isolation of dna mutants was important in several ways 1. It allowed for the identification of the proteins that were defective in the mutant 2. It allowed for the mapping of these mutations along the E. coli chromosome 3. It provided an important starting point for the subsequent cloning and sequencing of these genes 11-62 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

53 11.3 EUKARYOTIC DNA REPLICATION
The bacterial enzymes described in Table 1.1 have also been found in eukaryotes Nevertheless, DNA replication in eukaryotes is more complex Large linear chromosomes Tight packaging within nucleosomes More complicated cell cycle regulation 11-63 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

54 Multiple Origins of Replication
Eukaryotes have long linear chromosomes They therefore require multiple origins of replication To ensure that the DNA can be replicated in a reasonable time In 1968, Huberman and Riggs provided evidence for the multiple origins of replication Refer to Figure 11.19 DNA replication proceeds bidirectionally from many origins of replication Refer to Figure 11.20 11-64 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

55 Fig

56 Fig

57 Figure 11-14 Copyright © 2006 Pearson Prentice Hall, Inc.
Figure A demonstration of the multiple origins of replication along a eukaryotic chromosome. Each origin is apparent as a replication bubble along the axis of the chromosome. Arrows identify some of these replication bubbles. Figure Copyright © 2006 Pearson Prentice Hall, Inc.

58 Telomeres and DNA Replication
Linear eukaryotic chromosomes have telomeres at both ends The term telomere refers to the complex of telomeric DNA sequences and bound proteins 11-75 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

59 Telomeric sequences consist of
Moderately repetitive tandem arrays 3’ overhang that is nucleotides long Figure 11.23 Telomeric sequences typically consist of Several guanine nucleotides Often many thymine nucleotides Refer to Table 11.5 11-76 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

60 11-77

61 DNA polymerases possess two unusual features
1. They synthesize DNA only in the 5’ to 3’ direction 2. They cannot initiate DNA synthesis These two features pose a problem at the 3’ end of linear chromosomes Figure 11.24 11-78 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

62 Figure 11-16 Copyright © 2006 Pearson Prentice Hall, Inc.
Figure Diagram illustrating the difficulty encountered during the replication of the ends of linear chromosomes. A gap (- -b- -) is left following synthesis on the lagging strand. Figure Copyright © 2006 Pearson Prentice Hall, Inc.

63 Therefore if this problem is not solved
The linear chromosome becomes progressively shorter with each round of DNA replication Indeed, the cell solves this problem by adding DNA sequences to the ends of telomeres This requires a specialized mechanism catalyzed by the enzyme telomerase Telomerase contains protein and RNA The RNA is complementary to the DNA sequence found in the telomeric repeat This allows the telomerase to bind to the 3’ overhang The lengthening mechanism is outlined in Figure 11.25 11-79 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

64 The binding-polymerization-translocation cycle can occurs many times
Step 1 = Binding The binding-polymerization-translocation cycle can occurs many times Step 2 = Polymerization This greatly lengthens one of the strands Step 3 = Translocation The complementary strand is made by primase, DNA polymerase and ligase Figure 11.25 RNA primer 11-80

65 Figure 11-17 Copyright © 2006 Pearson Prentice Hall, Inc.
Figure The predicted solution to the problem posed in Figure 11–16. The enzyme telomerase directs synthesis of the TTGGGG sequences, resulting in the formation of a hairpin structure. The gap can now be filled, and, following cleavage of the hairpin structure, the process averts the creation of a gap during replication of the ends of linear chromosomes. Figure Copyright © 2006 Pearson Prentice Hall, Inc.


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