DNA Replication, Chapter 11 PDF

Because learning changes everything. ® Chapter 11 DNA Replication Genetics: Analysis & Principles EIGHTH EDITION Robert J. Brooker © McGraw Hill LLC. All rights reserved. No reproduction or distribution without the prior written consent of McGraw Hill LLC.  DNA Rep...

Because learning changes everything. ® Chapter 11 DNA Replication Genetics: Analysis & Principles EIGHTH EDITION Robert J. Brooker © McGraw Hill LLC. All rights reserved. No reproduction or distribution without the prior written consent of McGraw Hill LLC.  DNA Replication © McGraw Hill © Clive Freeman/The Royal Institution/Science Source 2  Introduction 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 sequences It occurs very quickly, very accurately and at the appropriate time in the life of the cell © McGraw Hill 3  11.1 Structural Overview of DNA Replication DNA replication relies on the complementarity of DNA strands A pairs with T and G pairs with C The AT/GC 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 4  (a) The mechanism of DNA replication Fig 11.1a © McGraw Hill 5  (b) The products of replication Fig 11.1b © McGraw Hill 6  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 This is the correct model Dispersive model Parental and daughter DNA segments are interspersed in both strands following replication © McGraw Hill 7  Original double helix First round of replication Second round of replication (a) Conservative model Fig 11.2a © McGraw Hill 8  Original double helix First round of replication Second round of replication (b) Semiconservative model (correct model) Fig 11.2b © McGraw Hill 9  Original double helix First round of replication Second round of replication (c) Dispersive Model Fig 11.2c © McGraw Hill 10  Experiment 11A: Which Model of DNA Replication is Correct? In 1958, Matthew Meselson and Franklin Stahl devised a method to investigate these models They were able to experimentally distinguish between daughter and parental strands using light and heavy nitrogen Hypothesis: Based on Watson’s and Crick’s ideas, the hypothesis was that DNA replication is semiconservative. © McGraw Hill 11  Testing the Hypothesis 1. Add an excess 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 15 N-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 12  Testing the Hypothesis 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 13  Fig 11.3 © McGraw Hill 14  The Data © McGraw Hill M. Meselson & F. Stahl (1958). “The replication of DNA in Eschericia coli.” PNAS, 44(7): 671-682, Fig 4A. Courtesy of M. Meselson 15  The Data After one generation, DNA is “half-heavy” Consistent with both semi-conservative and dispersive models After ~ two generations, DNA is of two types: Equal amounts of “light” and “half-heavy” Consistent with only the semi-conservative model © McGraw Hill 16  11.2 Bacterial DNA Replication: The Formation of Two Replication Forks at the Origin of 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 17  Bacterial chromosome replication Fig 11.4 © McGraw Hill 18  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 important DnaA boxes- sites for the binding of DnaA protein AT-rich regions- sites where the DNA strands separate GATC methylation sites- sites that help to regulate DNA replication © McGraw Hill 19  Fig 11.5 © McGraw Hill 20  Events that Occur at oriC DnaA proteins bind to DnaA boxes and to each other Additional proteins bind and cause the DNA to bend Strands separate at AT-rich region DnaB/helicase then binds to the origin Further separates the DNA strands Composed of six subunits Travels along the DNA in the 5’ to 3’ direction Uses energy from ATP Replication occurs in both directions – bidirectional replication © McGraw Hill 21  Fig 11.6 © McGraw Hill 22  GATC Methylation Sites in Replication GATC methylation sites are involved in regulating 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 prevented from occurring too soon © McGraw Hill 23  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 DNA gyrase, also called Topoisomerase II, 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 24  Synthesis of RNA Primers and DNA Then short RNA primers are synthesized by primase These short RNA strands start, or prime, DNA synthesis Typically, 10 to 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 25  Fig 11.7 © McGraw Hill 26  DNA Polymerases 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 27  DNA Polymerases DNA pol III Responsible for most of the DNA replication Composed of 10 different subunits (see Table 11.2 in your textbook) The ⍺ 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 28  Subunit Composition of DNA Polymerase III Holoenzyme from E. coli Subunit Composition of DNA polymerase III Holoenzyme from E. coli Subunit(s) Function α Synthesizes DNA ε 3' to 5’ exonuclease site that removes mismatched nucleotides (proofreading) θ Accessory protein that stimulates the proofreading function β Clamp protein, which allows DNA polymerase to slide along the DNA without falling off τ, γ, δ, δ′, ψ, Clamp loader complex, involved with helping the clamp and χ protein bind to the DNA Tab 11.2 © McGraw Hill 29  Structure of DNA Polymerase 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 30  (a) Schematic side view of DNA polymerase III Fig 11.8a © McGraw Hill 31  Features of DNA Polymerase DNA polymerases cannot initiate DNA synthesis by linking two individual nucleotides on a bare template strand 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 32  Fig 11.9 © McGraw Hill 33  Synthesis of Leading and Lagging Strands 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 34  Synthesis of Leading and Lagging Strands 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 to 200 in eukaryotes) These are termed Okazaki fragments after their discoverers © McGraw Hill 35  DNA Synthesis at the Replication Fork 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 covalent (ester) bond to connect the DNA backbones Thereby connecting the Okazaki fragments © McGraw Hill 36  Fig 11.10 © McGraw Hill 37  Fig 11.11 © McGraw Hill 38  DNA Replication Complexes DNA helicase and primase are physically bind 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 39  Fig 11.12 © McGraw Hill 40  Termination Sequences 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 © McGraw Hill 41  Termination of Replication In a given cell, only one ter sequence is required to stop one fork, and the other fork ends its DNA synthesis when it reaches the stopped fork DNA replication ends when oppositely advancing forks meet (usually at T1 or T2) Finally, DNA ligase covalently links the two daughter strands Fig 11.13 © McGraw Hill 42  Termination of Replication DNA replication often results in two intertwined molecules Intertwined circular molecules are termed catenanes These are separated by the action of DNA gyrase Fig 11.14 © McGraw Hill 43  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 I Various other proteins 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 44  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 temperature Figure 11.15 shows a general strategy for the isolation of ts mutants Fig 11.15 © McGraw Hill 45  Isolation of Mutants The mutants fell into two groups when shifted to the non- permissive temperature Some showed a rapid arrest These genes coded proteins needed for synthesis of the DNA Other mutants completed their current round of replication but could not start another Coded proteins needed for initiation of replication Table 11.3 (see your textbook) summarizes some of the genes that were identified using this strategy Include mutations in DNA polymerase III, primase, helicase, DnaA, DnaC © McGraw Hill 46  11.4 Bacterial DNA Replication: Chemistry and Accuracy DNA polymerase 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 47  Fig 11.16 © McGraw Hill 48  DNA Polymerase III is a Processive Enzyme 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 49  DNA Polymerase III is a Processive Enzyme 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 50  Fidelity 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 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 51  Fidelity Mechanisms 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 from 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 52  Fidelity Mechanisms Proofreading function of DNA polymerase DNA polymerase can identify a mismatched nucleotide and remove it from the daughter strand It 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 53 © McGraw Hill 54  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 proteins involved in bacterial DNA replication 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 55  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 56  Evidence for Multiple Origins of Replication 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 Fig 11.18 © McGraw Hill Courtesy of Dr. Joel A. Huberman 57  Replication of Eukaryotic Chromosomes Replication bubbles from multiple origins merge into completely replicated chromosomes Fig 11.19 © McGraw Hill 58  Eukaryotic Origins of Replication The origins of replication found in simple 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 Have a copy of the ARS consensus sequence and B1 and/or B2 sequences ATTTAT(A or G)TTTA B1 and B2 (enhance function of origin of replication) Separation of DNA occurs within B2 Are found within a nucleosome-free region (NFR) © McGraw Hill 59  Eukaryotic Origins of Replication The origins of replication in more complex eukaryotes Do not contain a consensus sequence analogous to the ARS consensus sequence Are more dynamic Contain G-rich sequences, or G4 motifs Form a G-quadraplex - four-stranded helical structure (Fig 11.20) The G4 motif in animals is found in a nucleosome-free region The flanking histone modifications favors an open conformation Promoters and CpG islands are frequently found in the NFR © McGraw Hill 60  General features of origins of replication in eukaryotes Access the text alternative for slide images. Fig 11.20 © McGraw Hill 61  Eukaryotic Origins of Replication Three main classes of replication origins in complex eukaryotes Constitutive: used all the time Flexible: used in a random manner; most common type Dormant: used during cell differentiation or only at a specific stage of development © McGraw Hill 62  Eukaryotic Replication 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 63  Fig 11.21 © McGraw Hill 64  Eukaryotes Contain Several Different DNA Polymerases Mammalian cells contain well over a dozen different DNA polymerases Refer to Table 11.4 (see your textbook) Four: alpha (⍺), delta (δ), epsilon (ε) and gamma (γ) have the primary function of replicating DNA ⍺, δ and ε  Nuclear DNA γ  Mitochondrial DNA © McGraw Hill 65  Functions of DNA Polymerases in Replication DNA pol ⍺ is the only polymerase to associate with primase The DNA pol ⍺/primase complex synthesizes a short RNA-DNA hybrid primer 10 RNA nucleotides followed by 20 to 30 DNA nucleotides The exchange of DNA pol ⍺ 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 66  Functions of DNA Polymerases in DNA Repair DNA polymerases play a role in DNA repair Many are translesion-replicating polymerases Involved in the replication of damaged DNA They can synthesize a complementary strand over the abnormal region © McGraw Hill 67  Flap Endonuclease Removes RNA Primers Distinct from prokaryotic replication Polymerase δ runs into primer of adjacent Okazaki fragment Pushes a portion of primer into short flap Flap endonuclease removes the primer If a flap is too long, it is cleaved by Dna2 nuclease/helicase Cuts long flap into short flap © McGraw Hill 68  Fig 11.22 © McGraw Hill 69  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 70  Telomeric DNA Sequences Telomeric sequences consist of moderately repetitive tandem arrays 3’ overhang that is 12 to 16 nucleotides long Telomeric sequences typically consist of Several guanine nucleotides Many thymine nucleotides Refer to Table 11.5 (see your textbook) Fig 11.23 © McGraw Hill 71  Replication Problem at the Ends of Linear Chromosomes DNA polymerases possess two unusual features They synthesize DNA only in the 5’ to 3’ direction They cannot initiate DNA synthesis on a bare (unprimed) DNA strand At the 3’ ends of linear chromosomes - the end of the strand cannot be replicated! Fig 11.24 © McGraw Hill 72  Replication Problem at the Ends of Linear Chromosomes If this problem is not solved The linear chromosome becomes progressively shorter with each round of DNA replication 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 73  Enzymatic Action of Telomerase Step 1: Binding Step 2: Polymerization Step 3: Translocation These steps are repeated many times to lengthen one strand © McGraw Hill 74  Fig 11.25 © McGraw Hill 75  Telomere Length in Cancer and Aging 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 76  Telomere Length in Cancer and Aging 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 77