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

INTRODUCTION DNA replication is the process by which the genetic material is copied It occurs very quickly, very accurately and at the appropriate time in the life of the cell This chapter examines how! 11-2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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

Identical base sequences Figure 11.1 11-4

Experiment 11A: Which Model of DNA Replication is Correct? In the late 1950s, three different mechanisms were proposed for the replication of DNA 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

Figure 11.2 11-6

11.2 BACTERIAL DNA 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

Figure 11.4 11-14

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

Figure 11.5 11-16

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

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

This generates positive supercoiling ahead of each replication fork DNA helicase separates the two DNA strands by breaking the hydrogen bonds between them This generates positive supercoiling ahead of each replication fork DNA gyrase travels ahead of the helicase and alleviates these supercoils Single-strand binding proteins bind to the separated DNA strands to keep them apart Then short (10 to 12 nucleotides) RNA primers are synthesized by DNA primase These short RNA strands start, or prime, DNA synthesis The leading strand has a single primer, the lagging strand needs multiple primers They are later removed and replaced with DNA 11-19 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-20 Figure 11.7 LEADING STRAND LOGGING STRAND Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-21 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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, II, III, IV and V DNA pol I and III Normal replication DNA pol II, IV and V DNA repair and replication of damaged DNA 11-22 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

DNA Polymerases DNA pol I DNA pol III Composed of a single polypeptide Removes the RNA primers and replaces them with DNA DNA pol III Responsible for most of the DNA replication Composed of 10 different subunits (Table 11.2) The a subunit synthesizes DNA The other 9 fulfill other functions The complex of all 10 is referred to as the DNA pol III holoenzyme 11-23 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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Bacterial DNA polymerases may vary in their subunit composition However, they have the same type of catalytic subunit Structure resembles a human right hand Template DNA thread through the palm; Thumb and fingers wrapped around the DNA Figure 11.8 11-25 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-26 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 11-26

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 11-27 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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 11-28 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 11.10 11-29

DNA polymerase III synthesizes base pairs at a rate of around 1000 nucleotides per second

Termination of Replication Opposite to oriC is a pair of termination sequences called ter sequences These are designated T1 and T2 T1 stops counterclockwise forks, T2 stops clockwise The protein tus (termination utilization substance) binds to these sequences It can then stop the movement of the replication forks Refer to Figure 11.12 11-34 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-35 Figure 11.12 Allows the advancement of the CW-moving fork Prevents the movement of the CCW-moving fork Allows the advancement of the CCW-moving fork Prevents the movement of the CW-moving fork 11-35 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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 11-36 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Catalyzed by DNA topoisomerases Figure 11.13 Catenanes Catalyzed by DNA topoisomerases 11-37

DNA Replication Complexes DNA helicase and primase are physically bound to each other to form a complex called the primosome This complex leads the way at the replication fork The primosome is physically associated with the DNA polymerase holoenzyme forming the replisome Figure 11.14 provides a three-dimensional view of DNA replication 11-38 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 11.14 11-39

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

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

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 Refer to figure 11.15 11-44 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-45 Figure 11.15 A schematic drawing of proofreading Site where DNA backbone is cut A schematic drawing of proofreading Figure 11.15 11-45

Bacterial DNA Replication is Coordinated with Cell Division Bacterial cells can divide into two daughter cells at an amazing rate E. coli  20 to 30 minutes Therefore it is critical that DNA replication take place only when a cell is about to divide Bacterial cells regulate the DNA replication process by controlling the initiation of replication at oriC E. coli does this via two different mechanisms Refer to Figures 11.16 and 11.17 11-46 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The amount of DnaA protein provides a way to regulate DNA replication Figure 11.16 11-47

11-48 Figure 11.17 DNA adenine methyltransferase Recognizes the 5’ – GATC – 3’ sequence and attaches a methyl group onto the adenine DNA adenine methyltransferase Methylation of GATC sites in oriC Figure 11.17 11-48

11-49 Figure 11.17 Methylation of GATC sites in oriC Before replication, GATC sites are methylated on both strands This full methylation facilitates initiation of DNA replication Following replication, GATC sites are not methylated on the daughter strands This half-methylation does not efficiently initiate replication Several minutes will pass before Dam methylase will methylate the GATC sites in the daughter strands Methylation of GATC sites in oriC Figure 11.17 11-49

11.3 EUKARYOTIC DNA REPLICATION Eukaryotic DNA replication is not as well understood as bacterial replication The two processes do have extensive similarities, The bacterial enzymes described in Table 11.1 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

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 11.20 DNA replication proceeds bidirectionally from many origins of replication Refer to Figure 11.21 11-64 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 11.21 11-65

Multiple Origins of Replication The origins of replication found in eukaryotes have some similarities to those of bacteria Origins of replication in Saccharomyces cerevisiae are termed ARS elements (Autonomously Replicating Sequence) They are 100-150 bp in length They have a high percentage of A and T They have three or four copies of a specific sequence Similar to the bacterial DnaA boxes 11-66 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Multiple Origins of Replication Replication begins with assembly of the prereplication complex (preRC) Consist of 14 different proteins. 6 of them called origin replication complex. An important part of this is the Origin recognition complex (ORC) A six-subunit complex that acts as the initiator of eukaryotic DNA replication DNA replication at the origin begins with the binding of ORC, which usually occurs during G1 phase. Other preRC proteins bind including MCM helicase 11-67 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

MCM is an acronym for minichromosome maintenance.

Binding of MCM completes DNA replication licensing The origin is capable of initiating DNA synthesis only those origins with MCM helicase can initiate DNA synthesis Binding of at least 22 additional proteins is required to initiate synthesis during S phase

Bénédicte Recolin et al 2014

Eukaryotes Contain Several Different DNA Polymerases Mammalian cells contain well over a dozen different DNA polymerases Refer to Table 11.4 Four: alpha (a), delta (d), epsilon (e) and gamma (g) have the primary function of replicating DNA a, d and e  Nuclear DNA g  Mitochondrial DNA 11-68 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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DNA pol a is the only polymerase to associate with primase The DNA pol a/primase complex synthesizes a short RNA-DNA hybrid 10 RNA nucleotides followed by 20 to 30 DNA nucleotides This is used by DNA pol d or e for the processive elongation of the leading and lagging strands Current evidence suggests a greater role for DNA pol d The exchange of DNA pol a for d or e is called a polymerase switch It occurs only after the RNA-DNA hybrid is made Refer to Figure 11.22 11-70 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 11.22 11-71

DNA polymerases also play a role in DNA repair DNA pol b is not involved in DNA replication It plays a role in base-excision repair Removal of incorrect bases from damaged DNA Recently, more DNA polymerases have been identified Lesion-replicating polymerases Involved in the replication of damaged DNA They can synthesize a complementary strand over the abnormal region 11-72 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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 11-73 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Telomeric sequences consist of Moderately repetitive tandem arrays TTAGG 3’ overhang that is 12-16 nucleotides long Figure 11.23 Telomeric sequences typically consist of Several guanine nucleotides Often many thymine nucleotides Refer to Table 11.5 11-74 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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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-the end of the strand cannot be replicated! Figure 11.24 11-76

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 11-77 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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 end is now lengthened Figure 11.25 RNA primer 11-78