1 It is the process by which the genetic material is copiedDNA replication It is the process by which the genetic material is copied The original DNA strands are used as templates for the synthesis of new strands It occurs very quickly, very accurately and at the appropriate time in the life of the cell Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

2 STRUCTURAL OVERVIEW OF DNA REPLICATIONDNA 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

3 A pairs with T and G with C to form new strandIdentical base sequences

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

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6 Their experiment can be summarized as suchIn 1958, Matthew Meselson and Franklin Stahl devised a method to investigate these models Their experiment can be summarized as such 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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

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

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10 BACTERIAL DNA REPLICATIONDNA 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 two replication forks eventually meet at the opposite side of the bacterial chromosome This ends replication Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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12 Initiation of ReplicationThe 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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14 DNA replication is initiated by the binding of DnaA proteins to the DnaA box sequencesThis binding stimulates the cooperative binding of additional ATP-bound DnaA proteins to form a large complex 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

15 Travels along the DNA in the 5’ to 3’ directionUses energy from ATP Bidirectional replication

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

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

18 DNA Polymerases DNA pol I Composed of a single polypeptideRemoves the RNA primers and replaces them with DNA Has 5’ to 3’ polymerase activity, 3’ to 5’ exonuclease activity and 5’ to 3’ exonuclease activity 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 Has 5’ to 3’ polymerase activity and 3’ to 5’ exonuclease activity Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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

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

21 Unusual features of DNA polymerase functionDNA 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

22 Leading strand Lagging strandThe 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

23 DNA pol I removes the RNA primers and fills the resulting gap with DNAIt 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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

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26 The Reaction of DNA PolymeraseDNA 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 In the process, the last two phosphates of the incoming nucleotide are released In the form of pyrophosphate (Ppi) Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

27 Innermost phosphate

28 DNA Polymerase III is a Processive EnzymeDNA polymerase III remains attached to the template as it is synthesizing the daughter strand This processive feature is due to several different subunits in the DNA pol III holoenzyme b subunit is in the shape of a ring It is termed the clamp protein g subunit is needed for b to initially clamp onto the DNA It is termed the clamp-loader protein d, d’ and y subunits are needed for the optimal function of the a and b subunits Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

29 Termination of ReplicationOpposite 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

30 Termination of ReplicationDNA 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

31 Helicase and Primase are linked together in a “primosome”Two DNA polymerase III holoenzymes and the primosome are linked together to form the Replisome

32 DNA Replication ComplexesTwo 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

33 DNA Replication ComplexesLagging 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

34 Proofreading MechanismsDNA 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

35 Proofreading Mechanisms1. 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

36 Proofreading Mechanisms3. 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

37 A schematic drawing of proofreadingSite where DNA backbone is cut A schematic drawing of proofreading

38 Bacterial DNA Replication is Coordinated with Cell DivisionBacterial cells can divide into two daughter cells at an amazing rate E. coli  20 to 30 minutes 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

39 DNA Replication Can Be Studied In VitroThe 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

40 EUKARYOTIC DNA REPLICATIONEukaryotic 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

41 Multiple Origins of ReplicationEukaryotes have long linear chromosomes They therefore require multiple origins of replication To ensure that the DNA can be replicated in a reasonable time DNA replication proceeds bidirectionally from many origins of replication Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

42 Chromosome Sister chromatids Origin Origin Origin Centromere OriginCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. © This article was published in Journal of Molecular Biology. Mar 14;32(2). Huberman JA. Riggs AD. “Links On the mechanism of DNA replication in mammalian chromosomes." Copyright Elsevier, Reprinted with permission. Chromosome Sister chromatids Origin Origin Origin Centromere Origin Origin Before S phase During S phase End of S phase

43 Multiple Origins of ReplicationThe 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 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

44 Multiple Origins of ReplicationReplication begins with assembly of the prereplication complex (preRC) Consists of at least 14 different proteins Binding of at least 22 additional proteins is required to initiate synthesis during S phase Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

45 Eukaryotes Contain Several Different DNA PolymerasesMammalian cells contain well over a dozen different DNA polymerases 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

46 DNA pol a is the only polymerase to associate with primaseThe 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 e or d for the processive elongation of the leading and lagging strands, respectively 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

47 Flap Endonuclease Removes RNA PrimersThis is function of Pol I in bacteria Polymerase d runs into primer of adjacent Okazaki fragment Pushes portion of primer into short flap Flap endonuclease removes the primer Long flaps are removed by Dna2 nuclease Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

48 5′ 3′ 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′ 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′ Process continues until the entire RNA primer is removed. DNA ligase seals the two fragments together. 5′ 3′ 3′ 5′ Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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

50 Telomeric sequences consist ofModerately repetitive tandem arrays 3’ overhang that is nucleotides long Telomeric sequences typically consist of Several guanine nucleotides Often many thymine nucleotides Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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52 DNA polymerases possess two unusual features1. 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!

53 Therefore if this problem is not solvedThe 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

54 Telomerase reverse transcriptase (TERT) activityStep 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 RNA primer