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Transcript of Principles of Biochemistry Fourth Edition Chapter 20 DNA Replication, Repair, and Recombination...
![Page 1: Principles of Biochemistry Fourth Edition Chapter 20 DNA Replication, Repair, and Recombination Copyright © 2006 Pearson Prentice Hall, Inc. Horton Moran.](https://reader035.fdocuments.net/reader035/viewer/2022081501/56649c7b5503460f9492faaf/html5/thumbnails/1.jpg)
Principles of BiochemistryFourth Edition
Chapter 20DNA Replication, Repair,
and Recombination
Copyright © 2006 Pearson Prentice Hall, Inc.
Horton • Moran • Scrimgeour • Perry • Rawn
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Chapter 20 - DNA Replication, Repair and Recombination
• Holliday junction, an intermediate formed during recombination between two double-stranded DNA molecules
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Fig 20.1
• Semiconservative DNA replication
• Each strand of DNA acts as a template for synthesis of a new strand
• Daughter DNA contains one parental and one newly synthesized strand
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20.1 Chromosomal DNA Replication Is Bidirectional
• E. coli chromosome is circular, double-stranded DNA (4.6x103 kilobase pairs, kb)
• Replication begins at a unique site (origin)
• Proceeds bidirectionally until the two replication complexes meet (termination site)
• Replisome - protein machinery for replication (one replisome at each of 2 replication forks)
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Fig 20.2 Bidirectional DNA replication in E. coli
• New strands of DNA are synthesized at the two replication forks where replisomes are located
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Fig 20.2 (cont)
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Fig. 20.3 Autoradiograph of a replicating E. coli chromosome
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Eukaryotic Replication
• Eukaryotic chromosomes are large linear, double-stranded DNA molecules
• Fruit fly large chromosomes ~5.0x104 kb (~10x larger than E. coli)
• Replication is bidirectional
• Multiple sites of initiation of DNA synthesis (versus one site in E. coli)
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Fig 20.4 Replicating DNA in the fruit fly
• Large number of replication forks at opposite ends of “bubbles” of duplicated DNA
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20.2 DNA Polymerase
• E. coli contains three DNA polymerases
• DNA polymerase I - repairs DNA and participates in DNA synthesis of one strand
• DNA polymerase II - role in DNA repair
• DNA polymerase III - the major DNA replication enzyme, responsible for chain elongation
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Fig 20.5 DNA Polymerase III: subunit composition
Holoenzyme
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A. Chain Elongation Is a Nucleotidyl-Group-Transfer Reaction
• Figure 20.6 (next 3 slides)
• Base pair between incoming deoxynucleotide 5’ triphosphate (blue) and a residue of the parental strand
• Terminal 3’ OH attacks -phosphorous of incoming nucleotide to form new phosphodiester linkage
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B. DNA Polymerase III Remains Bound to the Replication Fork
• DNA polymerase III is a processive enzyme (remains bound to the replication fork until replication is complete)
• -Subunits form a sliding clamp which surrounds the DNA molecule
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Fig 20.7 Sliding clamp of -subunits of DNA pol III
• Two -subunits associate to form a head-to-tail dimer in the shape of a ring that completely surrounds the DNA
• Remaining subunits of DNA pol III are bound to this structure
E coli archeabacteria bacteriophage T4
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Fig 20.8 Bacteriophage DNA polymerase bound to DNA
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C. Proofreading Corrects Polymerization Errors
• DNA polymerase III holoenzyme also possesses 3’ 5’ exonuclease activity
• Pol III can catalyze both chain elongation and degradation
• Recognizes distortion in the DNA caused by incorrectly paired bases
• Exonuclease activity removes mispaired nucleotide before polymerization continues
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20.3 DNA Polymerase Synthesizes Two Strands Simultaneously
• DNA pol III catalyzes chain elongation only in the 5’ 3’ direction (antiparallel DNA strands)
• Leading strand - synthesized by polymerization in the same direction as fork movement
• Lagging strand - synthesized by polymerization in the opposite direction of fork movement
• Two core complexes of DNA pol III, one for leading, one for lagging strand
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Fig 19.13
• Complementary base pairing and stacking in DNA
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Fig 20.9 Diagram of the replication fork
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A. Lagging-Strand Synthesis is Discontinuous
• Leading strand is synthesized as one continuous polynucleotide (beginning at origin and ending at the termination site)
• Lagging strand is synthesized discontinuously in short pieces (Okazaki fragments)
• Pieces of the lagging strand are then joined by a separate reaction
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Fig 20.10
• Demonstration of discontinuous DNA synthesis
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Fig 20.10
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Fig 20.10 (cont)
(From previous slide)
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B. Each Okazaki Fragment Begins with an RNA Primer
• Primosome is a complex containing primase enzyme which synthesizes short pieces of RNA at the replication fork (complementary to the lagging-strand template)
• DNA pol III uses the RNA primer to start the lagging-strand DNA synthesis
• Replisome - includes primosome, DNA pol III
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Fig 20.11 Diagram of lagging-strand synthesis
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C. Okazaki Fragments Are Joined by Action of DNA Polymerase I and DNA Ligase
• Okazaki fragments are joined to produce a continuous strand of DNA in 3 steps:
(1) Removal of the RNA primer
(2) Synthesis of replacement DNA
(3) Sealing of adjacent DNA fragments
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Fig 20.12
• Klenow (large) fragment of DNA pol I, lacks 3’-5’ exonuclease activity
• Used for DNA synthesis
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DNA Polymerase I Activities
• The 5’ 3’ activity of DNA pol I removes the RNA primer at the beginning of each Okazaki fragment
• Synthesizes nick translation: polymerase activity synthesizes DNA in place of RNA
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DNA Ligase Activity
• Catalyzes the formation of a phosphodiester linkage between 3’-hydroxyl and 5’-phosphate of adjacent Okazaki fragments
• Eukaryotic enzymes require ATP cosubstrate
• E. coli DNA ligase uses NAD+ as a cosubstrate
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Fig 20.13 Joining of Okazaki fragments by DNA pol I and DNA ligase
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Fig 20.13 (continued)
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Fig 20.13 (continued)
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Fig 20.13 (continued)
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Fig 20.14 Mechanism of DNA ligase in E. coli
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20.4 Model of the Replisome
• Replisome contains: a primosome, DNA polymerase III holoenzyme, additional proteins
• DnaB helicase is part of the primosome and facilitates unwinding of the DNA helix
• Topoisomerases relieve supercoiling ahead of the replicating fork (not part of the replisome)
• Single-stranded binding proteins (SSBs) stabilize single-stranded DNA
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Fig 20.15 Replisome DNA synthesis
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Fig 20.15 (continued)
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Fig 20.15 (continued)
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Fig 20.15 (continued)
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AnimationsAnimations
• DNA Elongation
• Leading and Lagging Strand Synthesis
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20.5 Initiation and Termination of DNA Replication
• Replisome assembles at origin site (oriC)
• Initial assembly depends on unwinding of the DNA caused by binding certain proteins
• DnaA is one initiation protein
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Termination site (ter)
• Terminator utilization substance (Tus) binds to the ter site
• Tus inhibits helicase activity and thus prevents replication forks continuing through this region
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Fig 20.16 Origin (oriC) and terminus (ter) of DNA replication in E. coli
• dnaA is gene for protein DnaA (required to initiate replication)
• Red arrows indicate direction of movement of replication forks
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Fig 20.17 E. coli Tus bound to DNA
• Tus binds to specific sequences at the termination site of DNA replication
• Tus blocks movement of the replisome by inhibiting the helicase activity of the replisome.
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Box 20.1 Sequencing DNA Using Dideoxynucleotides
• Sanger method uses 2’,3’-dideoxynucleoside triphosphates (ddNTPs) which are incorporated at the 3’ end of a growing chain in place of a dNTP
• Since ddNTPs lack a 3’-hydroxyl group, subsequent nucleotide addition cannot take place
• Small amounts of ddNTP’s terminate replication of some chains at each step, leaving a set of fragments of different lengths
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Chemical structure of a ddNTP
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Box 20.1 Sanger method for sequencing
DNA
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Sanger method for sequencing DNA
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Sanger method (continued)
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Sanger method (continued)
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Automated Dideoxy DNA Sequencing with Fluorescent ddNTPs
(http://www.mun.ca/biology/scarr/Fluorescent_didoxy_sequencing.html)
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20.6 DNA Replication in Eukaryotes
• Mechanism similar to that in prokaryotes: leading strand continuous synthesis, lagging strand discontinuous synthesis
• Replication forks move more slowly, but many replication forks
• Okazaki fragments are shorter in eukaryotes (~100-200 residues)
• At least 5 different DNA polymerases
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Fig 20.18 Eukaryotic cell division cycle
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Table 20.2
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Accessory Proteins Associated with the Replication Fork
• PCNA (proliferating cell nuclear antigen) forms structure resembling -subunit sliding clamp (E. coli DNA polymerase III)
• RPC (replication factor C) similar to complex of DNA pol III
• RPA (replication factor A) similar to prokaryotic SSB
• Helicases also present to unwind DNA
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AnimationAnimation
• DNA Replication
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20.7 Repair of Damaged DNA
• DNA is the only cellular macromolecule that can be repaired
• DNA damage includes: base modificationsnucleotide deletions or insertionscross-linking of DNA strandsbreakage of phosphodiester backbone
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DNA Repair Mechanisms
• Specific repair enzymes scan DNA to detect any alterations
• Lesions may be fixed by direct repair, which does not require breaking the phosphodiester backbone of DNA
• Repair protects individual cells and subsequent generations
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A. Repair after Photodimerization:
An Example of Direct Repair
• Double-helical DNA is very sensitive to damage by ultaviolet (UV) light
• Dimerization of adjacent pyrimidines in a DNA strand is common (e.g. thymines)
• Replication cannot proceed in the presence of pyrimidine dimers (distort the template strand)
• Thymine dimers are repaired in all organisms
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Fig 20.19 Photodimerization of adjacent thymines
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Fig 20.20
• Repair of thymine dimers by DNA photolyase
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Fig 20.20
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Fig 20.20 (cont)
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Fig 20.20 (cont)
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B. Excision Repair
• DNA can be damaged by alkylation, methylation, deamination, loss of heterocyclic bases (depurination or depyrimidization)
• General excision-repair pathway can repair many of these defects
• Overall pathway is similar in all organisms
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Fig 20.21
• General excision-repair pathway
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Fig 20.21
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Fig 20.21 (cont)
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Fig 20.22 Hydrolytic deamination of cytosine to uracil
• Uracil in place of cytosine causes incorporation of an incorrect base during replication
• DNA glycosylases hydrolyze base-sugar N-glycosidic bonds
• Deaminated bases are then removed and replaced
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Fig 20.23 Uracil N-glycosylase (human mitochondria)
• Enzyme is bound to a uracil-containing nucleotide (green) that has been flipped out of the stacked region of DNA
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Fig 20.24
• Repair of damage from deamination of cytosine
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Fig 20.24
(AP site: apurine and apyrimidine
sites)
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Fig 20.24 (cont)
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Fig 20.24 (cont)
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20.8 Homologous Recombination
• Recombination - exchange or transfer of pieces of DNA from one chromosome to another or within a chromosome
• Homologous recombination - occurs between pieces of DNA that have closely related sequences
• Nonhomologous recombination occurs between unrelated sequences (e.g. Transposons )
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A. The Holliday Model of General Recombination
Figure 20.25
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Figure 20.25
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Fig 20.25 (cont)
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B. Recombination in E. coli
• Recombination starts with generation of single-stranded DNA with a free 3’ end
• In E. coli, RecBCD endonuclease binds to DNA, cleaves one strand, then unwinds DNA in an ATP-dependent reaction
• Strand exchange begins when single-stranded DNA invades a neighboring double helix
• Rec A is a strand exchange protein
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Fig 20.26
• Strand exchange catalyzed by RecA
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Fig 20.26
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Fig 20.26(cont)
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C. Recombination Can Be a Form of Repair
• Recombination enzymes probably evolved to help DNA repair (confers a selective advantage)
• Recombination also creates new combinations of genes on a chromosome, increasing chances of evolutionary survival
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Fig 20.27
• Action of Ruv proteins at Holliday junctions
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Fig 20.28 Model of RuvA and RuvB bound to Holliday junction
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Fig 20.29 Branch migration and resolution
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Box 20.2 Molecular Links Between DNA Repair and Breast Cancer
• Some cancers are due to mutations in the genes BRCA1 or BRCA2
• BRCA1 and BRCA2 are required for normal recombinational repair of double strand breaks (DSB)
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Fig 20.20 PCNA (proliferating cell nuclear antigen)
• Trimeric protein that forms sliding clamp that surrounds DNA
• Part of eukaryotic replisome
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Fig 20.28 Stereo view of a Holliday junction structure