Lecture 12 DNA Replication(2).pdf

Genetics Analysis & Principles Sixth Edition Chapter 11 DNA Replication © McGraw-Hill Education. All rights reserved. Authorized only for instru...

Genetics Analysis & Principles Sixth Edition Chapter 11 DNA Replication © McGraw-Hill Education. All rights reserved. Authorized only for instructor use in the classroom. No reproduction or further distribution permitted without the prior written consent of McGraw-Hill Education. DNA Replication © Clive Freeman/The Royal Institution/Science Source © McGraw-Hill Education. 11-2 Introduction (1 of 2) DNA replication is the process by which the genetic material is copied  The original DNA strands are used as templates for the synthesis of new strands with identical sequence It occurs very quickly, very accurately and at the appropriate time in the life of the cell  This chapter examines how! © McGraw-Hill Education. 11-3 Introduction (2 of 2) (b) The products of replication © McGraw-Hill Education. 11-4 11.1 Structural Overview of DNA Replication (1 of 2) DNA replication relies on the complementarity of DNA strands  A pairs with T and G pairs with C  The AT/GC rule or Chargaff’s rule The process is:  The two complementary DNA strands come apart  Each serves as a template strand for the synthesis of new complementary DNA strands  The two newly-made DNA strands = daughter strands  The two original DNA strands = parental strands © McGraw-Hill Education. 11-5 11.1 Structural Overview of DNA Replication (2 of 2) (a) The mechanism of DNA Replication © McGraw-Hill Education. 11-6 Experiment 11A: Which Model of DNA Replication is Correct? (1 of 3) 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 segments are interspersed in both strands following replication © McGraw-Hill Education. 11-7 Experiment 11A: Which Model of DNA Replication is Correct? (2 of 3) In 1958, Matthew Meselson and Franklin Stahl devised a method to investigate these models  They found a way to experimentally distinguish between daughter and parental strands Hypothesis: Based on Watson’s and Crick’s ideas, the hypothesis was that DNA replication is semiconservative. © McGraw-Hill Education. 11-8 Experiment 11A: Which Model of DNA Replication is Correct? (3 of 3) Experimental summary:  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 © McGraw-Hill Education. 11-9  Three Possible Models for DNA Replication (Figure 11.2) (1 of 3) (a) Conservative model © McGraw-Hill Education. 11-10  Three Possible Models for DNA Replication (Figure 11.2) (2 of 3) (b) Semiconservative model (correct model) © McGraw-Hill Education. 11-11  Three Possible Models for DNA Replication (Figure 11.2) (3 of 3) (c) Dispersive Model © McGraw-Hill Education. 11-12 Testing the Hypothesis (Figure 11.3) (1 of 3) 1. Add an access of 14N-containing compounds to the growth medium so all of the newly made DNA will contain 14N. 2. Incubate the cells for various lengths of time. Note: The 15N- labeled DNA is shown in purple and the 14N-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. © McGraw-Hill Education. 11-13 Testing the Hypothesis (Figure 11.3) (2 of 3) 4. Load a sample of the lysate onto a CsCl gradient. (Note: The average density of DNA is around 1.7 g/cm3, which is well isolated from other cellular molecules.) 5. Centrifuge the gradients until the DNA molecules reach their equilibrium densities. 6. DNA within the gradient can be observed under a UV light. © McGraw-Hill Education. 11-14 Testing the Hypothesis (Figure 11.3) (3 of 3) © McGraw-Hill Education. 11-15 The Data (1 of 2) M. Meselson & F. Stahl (1958). “The replication of DNA in Eschericia coli.” PNAS, 44(7): 671-682, Fig 4A. Courtesy of M. Meselson © McGraw-Hill Education. 11-16 The Data (2 of 2) After one generation, DNA is “half-heavy”  consistent with both semi-conservative and dispersive models After ~ two generations, DNA is of two types: “light” and “half-heavy”  consistent with only the semi-conservative model © McGraw-Hill Education. 11-17 11.2 Bacterial DNA Replication Bacterial DNA Replication: The formation of two replication forks at the origin of replication DNA synthesis begins at a site termed the origin of replication  Each bacterial chromosome has only one origin of replication 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 © McGraw-Hill Education. 11-18  Bacterial Chromosome Replication (1 of 2) (a) Bacterial chromosome replication © McGraw-Hill Education. 11-19 Bacterial Chromosome Replication (2 of 2) (b) Autoradiograph of an E. coli in the act of replication (b) Cold Spring Harber Symposia on Quantitative Biology (1963). 28: 43. John Cairns, © Cold Spring Harbor Laboratory Press © McGraw-Hill Education. 11-20 Initiation of Replication (1 of 2) 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 Figure 11.5 © McGraw-Hill Education. 11-21 Initiation of Replication (2 of 2) © McGraw-Hill Education. 11-22  Events that Occur at oriC (Figure 11.6) (1 of 2) DnaA proteins bind to DnaA boxes and to each other  Additional proteins bind to bend DNA  Strands separate at AT-rich region DnaB/helicase  Composed of six subunits  Travels along the DNA in the 5’ to 3’ direction  Uses energy from ATP © McGraw-Hill Education. 11-23 Events that Occur at oriC (Figure 11.6) (2 of 2) © McGraw-Hill Education. 11-24 GATC Methylation Sites in Replication GATC methylation sites are involved in regulating replication  Need to ensure only one round of replication DNA adenine methyltransferase (Dam) methylates the A on both strands  Immediately after replication, the daughter strand is not methylated  Takes several minutes to become methylated Initiation of replication only occurs efficiently on fully methylated DNA  Second round initiation is blocked © McGraw-Hill Education. 11-25 11.3 Bacterial DNA Replication: Synthesis of New DNA strands Unwinding of the Helix: DNA helicase separates the two DNA strands by breaking the hydrogen bonds between them This generates positive supercoiling ahead of each replication fork  Topoisomerase II also called 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  Bases are exposed and can hydrogen bond with individual nucleotides © McGraw-Hill Education. 11-26 Synthesis of RNA Primers and DNA Then short (10 to 12 nucleotides) RNA primers are synthesized by primase  These short RNA strands start, or prime, DNA synthesis Typically 10-12 nucleotides in length The leading strand has a single primer The lagging strand needs multiple primers They are eventually removed and replaced with DNA DNA polymerase enzymes are responsible for synthesizing the DNA © McGraw-Hill Education. 11-27 Proteins in Bacterial DNA Replication (Figure 11.7) (1 of 3) Functions of key proteins involved with bacterial DNA replication  DNA Helicase breaks the hydrogen bonds between the DNA strands.  Topoisomerase II alleviates positive supercoiling.  Single-stranded binding proteins keep the parental strands apart  Primase synthesizes an RNA primer © McGraw-Hill Education. 11-28 Proteins in Bacterial DNA Replication (Figure 11.7) (2 of 3)  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 © McGraw-Hill Education. 11-29 Proteins in Bacterial DNA Replication (Figure 11.7) (3 of 3) © McGraw-Hill Education. 11-30 DNA Polymerases (1 of 2) DNA polymerases are the enzymes that catalyze the attachment of nucleotides to synthesize a new DNA strand 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 © McGraw-Hill Education. 11-31 DNA Polymerases (2 of 2) DNA pol III  Responsible for most of the DNA replication  Composed of 10 different subunits (Table 11.2) The α subunit catalyzes bond formation between adjacent nucleotides (DNA synthesis) The other 9 fulfill other functions  The complex of all 10 subunits is referred to as the DNA pol III holoenzyme DNA pol I  Composed of a single polypeptide  Removes the RNA primers and replaces them with DNA © McGraw-Hill Education. 11-32 Subunit Composition of DNA Polymerase III Holoenzyme from E. coli TABLE 11.2 Subunit Composition of DNA polymerase III Holoenzyme from E. coli Subunit(s) Function α Synthesizes DNA 3' to 5' proofreading (removes mismatched ε nucleotides) Accessory protein that stimulates the proofreading θ function Clamp protein, which allows DNA polymerase to slide β along the DNA without falling off τ, γ, δ, δ′, ψ, Clamp loader complex, involved with helping the and χ clamp protein bind to the DNA © McGraw-Hill Education. 11-33 Structure of DNA Polymerase (1 of 2) Structure resembles a human right hand  Template DNA is threaded through the palm;  Thumb and fingers wrapped around the DNA Bacterial DNA polymerases may vary in their subunit composition  However, they all have a similar catalytic subunit © McGraw-Hill Education. 11-34 Structure of DNA Polymerase (2 of 2) (a) Schematic side view of DNA polymerase III © McGraw-Hill Education. 11-35 Features of DNA Polymerase (Figure 11.9) (1 of 2) DNA polymerases cannot initiate DNA synthesis by linking two individual nucleotides  Problem is overcome by the RNA primers synthesized by primase DNA polymerases can attach nucleotides only in the 5’ to 3’ direction, but the two strands are anti-parallel and go in opposite directions  Problem is overcome by synthesizing the new strands both toward, and away from, the replication fork © McGraw-Hill Education. 11-36 Features of DNA Polymerase (Figure 11.9) (2 of 2) © McGraw-Hill Education. 11-37 Synthesis of Leading and Lagging Strands (1 of 2) The two new daughter strands are synthesized in different ways and opposite orientations 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 © McGraw-Hill Education. 11-38 Synthesis of Leading and Lagging Strands (2 of 2) 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 in bacteria, 100-200 in eukaryotes) These are termed Okazaki fragments after their discoverers © McGraw-Hill Education. 11-39 DNA Synthesis at the Replication Fork (Figure 11.10) (1 of 2)  DNA pol I removes the RNA primers and fills the resulting gap with DNA  It uses a 5’ to 3’ exonuclease activity to digest the RNA and 5’ to 3’ polymerase activity to replace it with DNA  After the gap is filled a covalent bond is still missing  DNA ligase catalyzes the formation of a phosphodiester bond  Thereby connecting the DNA fragments © McGraw-Hill Education. 11-40 DNA Synthesis at the Replication Fork (Figure 11.10) (2 of 2) © McGraw-Hill Education. 11-41 Synthesis of Leading and Lagging Strands from a Single Origin of Replication (Figure 11.11) Figure 11.11 © McGraw-Hill Education. 11-42 DNA Replication Complexes (1 of 2) DNA helicase and primase are physically bound to each other to form a complex called the primosome  This complex better coordinates the actions of helicase and primase The primosome is physically associated with two DNA polymerase holoenzymes to form the replisome © McGraw-Hill Education. 11-43 DNA Replication Complexes (2 of 2) 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 © McGraw-Hill Education. 11-44  Lagging Strand Synthesis The lagging strand is looped  This allows the attached DNA polymerase to synthesize the Okazaki fragments in the normal 5’ to 3’ direction  The polymerase synthesizing the lagging strand is also moving toward the replication fork Upon completion of an Okazaki fragment, the enzyme releases the lagging template strand  The clamp loader complex then reloads the polymerase at the next RNA primer  Another loop is then formed This processed is repeated over and over again Refer to Figure 11.12 © McGraw-Hill Education. 11-45 Lagging Strand Synthesis (Figure 11.12) © McGraw-Hill Education. 11-46 Termination of Replication On the opposite side of the chromosome to oriC is a pair of termination sequences called ter sequences  These are designated T1 and T2 T1 stops counterclockwise forks, T2 stops clockwise forks The protein tus (termination utilization substance) binds to the ter sequences  tus bound to the ter sequences stops the movement of the replication forks Refer to Figure 11.13 © McGraw-Hill Education. 11-47 Termination of Replication (Figure 11.13) (1 of 2) Tus proteins binds ter sites to stop replication forks T1 prevents the advancement of the fork moving left to right (clockwise) T2 prevents the advancement of the fork that is moving right to left (counterclockwise) © McGraw-Hill Education. 11-48  Termination of Replication (Figure 11.13) (2 of 2) DNA replication ends when oppositely advancing forks meet (usually at T1 or T2) Finally DNA ligase covalently links the two daughter strands DNA replication often results in two intertwined molecules  Intertwined circular molecules are termed catenanes  These are separated by the action of topoisomeras © McGraw-Hill Education. 11-49 Isolation of Mutants (1 of 4) The isolation of mutants has been crucial in elucidating DNA replication  For example, mutants played key roles in the discovery of DNA pol I 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 © McGraw-Hill Education. 11-50 Isolation of Mutants (2 of 4) 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 temperature Figure 11.15 shows a general strategy for the isolation of ts mutants © McGraw-Hill Education. 11-51 Isolation of Mutants (3 of 4) 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 © McGraw-Hill Education. 11-52 Isolation of Mutants (4 of 4) The dna mutants fell into two groups when shifted to the non- permissive temperature  Some showed a rapid arrest These genes encoded enzymes needed for replication of the DNA  Other mutants completed their current round of replication but could not start another Encoded genes needed for initiation of replication Table 11.3 summarizes some of the genes that were identified using this strategy  Include mutations in DNA polymerase III, primase, helicase, DnaA, DnaC © McGraw-Hill Education. 11-53 11.4 Bacterial DNA Replication: Chemistry and Accuracy DNA polymerases catalyzes the formation of a covalent (ester) bond between the  Innermost phosphate group of the incoming deoxyribonucleoside 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)  Refer to Figure 11.16 © McGraw-Hill Education. 11-54 The Enzymatic Action of DNA polymerase (Figure 11.16) © McGraw-Hill Education. 11-55 DNA Polymerase III is a Processive Enzyme (1 of 2) DNA 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  β subunit forms a dimer in the shape of a ring around template DNA It is termed the clamp protein Once bound, the β subunits can freely slide along dsDNA Promotes association of holoenzyme with DNA  Complex of several subunits functions as a clamp loader © McGraw-Hill Education. 11-56 DNA Polymerase III is a Processive Enzyme (2 of 2) The effect of processivity is quite remarkable  In the absence of the β subunit  DNA pol III falls off the DNA template after about 10 nucleotides have been polymerized  Its rate is ~ 20 nucleotides per second  In the presence of the β subunit  DNA pol III stays on the DNA template long enough to polymerize up to 500,000 nucleotides  Its rate is ~ 750 nucleotides per second © McGraw-Hill Education. 11-57  Fidelity Mechanisms (1 of 4) 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  Stability of base pairing  Structure of the DNA polymerase active site  Proofreading function of DNA polymerase © McGraw-Hill Education. 11-58 Fidelity Mechanisms (2 of 4) Stability of proper base pairs  Complementary base pairs have much higher stability than mismatched pairs  Stability of base pairs only accounts for part of the fidelity Error rate for mismatched base pairs is 1 per 1,000 nucleotides Configuration of the DNA polymerase active site  Helix distortion caused by mispairing prevents incorrect nucleotide fitting properly in active site  This induced-fit phenomenon decreases the error rate to a range of 1 in 100,000 to 1 million © McGraw-Hill Education. 11-59 Fidelity Mechanisms (3 of 4) Proofreading function of DNA polymerase  DNA polymerases can identify a mismatched nucleotide and remove it from the daughter strand  The enzyme uses a 3’ to 5’ exonuclease activity to digest the newly made strand until the mismatched nucleotide is removed  DNA synthesis then resumes in the 5’ to 3’ direction © McGraw-Hill Education. 11-60 Fidelity Mechanisms (4 of 4) © McGraw-Hill Education. 11-61 11.5 Eukaryotic DNA Replication Eukaryotic DNA replication is not as well understood as bacterial replication  The two processes do have extensive similarities, The types of bacterial enzymes described in Table 11.1 have also been found in eukaryotes  Nevertheless, DNA replication in eukaryotes is more complex Large linear chromosomes Chromatin is tightly packed within nucleosomes More complicated cell cycle regulation © McGraw-Hill Education. 11-62 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 amount of time In 1968, Huberman and Riggs provided evidence for multiple origins of replication  Refer to Figure 11.18 DNA replication proceeds bidirectionally from many origins of replication  Refer to Figure 11.19 © McGraw-Hill Education. 11-63 Evidence for Multiple Origins of Replication (Figure 11.18) A pulse of radiolabeled nucleoside was taken up by cells and incorporated into newly made DNA strands. Labeled areas were interspersed between nonlabeled, indicating multiple regions had initiated replication Courtesy of Dr. Joel A. Huberman © McGraw-Hill Education. 11-64  Replication of Eukaryotic Chromosomes (Figure 11.19) Replication bubbles from multiple origins merge into completely replicated chromosomes © McGraw-Hill Education. 11-65 Eukaryotic 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 about 50 bp in length They have a high percentage of A and T ARS consensus sequence (ACS) ATTTAT(A or G)TTTA  Origins in more complex eukaryotes are not fully defined Many occur at sites defined by chromatin structure, not sequence © McGraw-Hill Education. 11-66 Eukaryotic Replication (Figure 11.20) (1 of 2) Replication begins with assembly of the prereplication complex (preRC) Includes the Origin recognition complex (ORC)  A six-subunit complex that acts as the first initiator of eukaryotic DNA replication Other preRC proteins include MCM Helicase  Binding of MCM completes DNA replication licensing  These origins are able to begin DNA synthesis © McGraw-Hill Education. 11-67 Eukaryotic Replication (Figure 11.20) (2 of 2) © McGraw-Hill Education. 11-68  Eukaryotes Contain Several Different DNA Polymerases Mammalian cells contain well over a dozen different DNA polymerases  Refer to Table 11.4 Four: alpha (α), delta (δ), epsilon (ε) and gamma (γ) have the primary function of replicating DNA  α, δ and ε  Nuclear DNA  γ  Mitochondrial DNA © McGraw-Hill Education. 11-69 Functions of DNA Polymerases in Replication DNA pol a is the only polymerase to associate with primase  The DNA pol α/primase complex synthesizes a short RNA-DNA hybrid primer 10 RNA nucleotides followed by 20 to 30 DNA nucleotides The exchange of DNA pol α for ε or δ is required for elongation of the leading and lagging strands.  This is called a polymerase switch  DNA pol ε is used for the processive elongation of the leading strand  DNA pol δ is used for the lagging strands © McGraw-Hill Education. 11-70  Functions of DNA Polymerases in DNA Repair DNA polymerases also play a role in DNA repair  DNA pol β is not involved in DNA replication  It plays a role in removal of incorrect bases from damaged DNA Recently, more DNA polymerases have been identified  Many are translesion-replicating polymerases Involved in the replication of damaged DNA They can synthesize a complementary strand over the abnormal region © McGraw-Hill Education. 11-71 Flap Endonuclease Removes RNA Primers (1 of 2) Distinct from prokaryotic replication Polymerase δ 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/helicase  Cuts long flap into short flap Refer to Figure 11.21 © McGraw-Hill Education. 11-72 Flap Endonuclease Removes RNA Primers (2 of 2) © McGraw-Hill Education. 11-73 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 © McGraw-Hill Education. 11-74 Telomeric DNA Sequences Telomeric sequences consist of moderately repetitive tandem arrays  3’ overhang that is 12-16 nucleotides long Telomeric sequences typically consist of  Several guanine nucleotides  Many thymine nucleotides Refer to Table 11.5 © McGraw-Hill Education. 11-75 Replication Problem at the Ends of Linear Chromosomes (1 of 2) DNA polymerases possess two unusual features  They synthesize DNA only in the 5’ to 3’ direction  They cannot initiate DNA synthesis At the 3’ ends of linear chromosomes - the end of the strand cannot be replicated! © McGraw-Hill Education. 11-76  Replication Problem at the Ends of Linear Chromosomes (2 of 2) 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 11.24 © McGraw-Hill Education. 11-77 Enzymatic Action of Telomerase (Figure 11.24) (1 of 2) Step 1: binding Step 2: polymerization Step 3: translocation These steps are repeated many times to lengthen one strand © McGraw-Hill Education. 11-78 Enzymatic Action of Telomerase (Figure 11.24) (2 of 2) © McGraw-Hill Education. 11-79 Telomere Length and Cancer (1 of 2) Telomeres tend to shorten in actively dividing cells  Telomere DNA is about 8,000 bp at birth  Can shorten to 1,500 in elderly person Cells become senescent when telomeres are short  Lose their ability to divide  Insertion of highly active telomerase can block senescence © McGraw-Hill Education. 11-80 Telomere Length and Cancer (2 of 2) Cancer cells commonly carry mutations increasing activity of telomerase  Prevents telomere shortening and senescence  May be a target for anti-cancer drug treatments © McGraw-Hill Education. 11-81  End of Presentation © McGraw-Hill Education. All rights reserved. Authorized only for instructor use in the classroom. No 11-82 reproduction or further distribution permitted without the prior written consent of McGraw-Hill Education.