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CHAPTER 27: DNA STRUCTURE, REPLICATION, REPAIR LECTURE TOPICS 1) DNA STRUCTURE Æ Models vs X-ray Æ Static vs Dynamic 2) DNA-PROTEIN INTERACTIONS Æ Sequence-specific vs non-specific 3) DNA TOPOISOMERASES – Changes in state of DNA Æ Cutting and sealing strands 4) DNA REPLICATION Æ E. coli chromosome Æ The players and the process 5) DNA RECOMBINATION 6) DNA MUTATIONS AND REPAIR E.coli is: Mr. DNA replication Mr. Transcription Mr. Translation Mr. Control of gene expression The Boss The E. is Escherichia, but you can call me Ed Models vs Real DNA structure from x-ray diffraction Watson-Crick Model “Real” B-DNA structure 36o turn/ base pair 28-42 turn/base pair Paired bases in same plane Propeller twisting (bases) Adjacent base pairs parallel Base roll (bends DNA) Structure is regular and not dependent on base sequence -Structure details are sequence specific (dependent) - sequence provides unique 3-D fit for protein-DNA interactions REAL B-DNA from X-ray structure BENDING OF DNA-B DNA: Bases are not in a plane PROPELLER TWISTING REAL B-DNA from X-ray structure Show Movie CHAPTER 27: DNA STRUCTURE, REPLICATION, REPAIR LECTURE TOPICS 1) DNA STRUCTURE Æ Models vs X-ray Æ Static vs Dynamic 2) DNA-PROTEIN INTERACTIONS Æ Sequence-specific vs non-specific 3) DNA TOPOISOMERASES – Changes in state of DNA Æ Cutting and sealing strands 4) DNA REPLICATION Æ E. coli chromosome Æ The players and the process 5) DNA RECOMBINATION 6) DNA MUTATIONS AND REPAIR Base pairs: H-bonding properties D A A A D A A A A A:T A D G:C Bases are H-donors (D) or acceptors (A) B-DNA A-DNA (and RNA) Up Down RNA 2`OH Steric Hindrance 3`Æ5` phosphodiester bond DNA DNA-RNA or (RNA-RNA) Z – DNA: exists but Function unknown Z B A DNA-RNA RNA-RNA Most DNA RARE Table 27-1 frp,, 5th DNA-Protein Interactions (DNA and/or RNA) [A key concept for rest of the course] - Non-specific [DNA sequence independent] - Specific [ DNA Sequence matters!!] Non-specific interactions: Deoxyribonuclease I _ Major Groove _ _ Arg / Lys have (+) charge in protein Minor Groove _ + _ (DNase I) + _ _ _ Sugar – Phospate backbone (-)charge EcoRV restriction enzyme recognition site Sequence-specific interactions 2-fold symmetry !! Asymmetrical DNA Recognition Site!! [Fig.9-37] EcoRV GATATC DNA bases form specific H-bonds with loop of EcoRV protein β-turn CTATAG Opens 500 Induced Fit DNA Sequence specific Interactions in DNA major groove EcoRV bends (kinks) DNA by 500 250 [Fig.9-40] 250 EcoRV β-turn loops H-bond with DNA Specific H-bonds in each EcoRV monomer * * * * * [Fig.9-39] * * * * * * Evolution: DNA sequence elements are conserved in active sites of some Type II restriction enzymes EcoRI recognition site GAATTC • CTTAAG l l l l l l Each DNA strand forms 6 H-bonds with Glu and Arg residues of Eco RI the enzyme. • • A total of 12 H-bonds form in Enzyme-DNA complex. EcoRI - DNA complex One side ~ Half a helix turn Top Two kinds of EcoRI-DNA Interactions DNA (+) dipole-phosphate backbone (-) interactions (at a specific location) Protein Arg specific H-bonds G base Circular DNA problem: How are ends of linear DNA joined to form circular DNA? Solution: (1967) DNA ligase was discovered Ligase was first in a NEW CLASS of enzymes called DNA Topoisomerases. These enzymes change DNA topology. [demonstrate with model] • Ligase requires a “nick”(break) in a 3’-5’ phosphodiester bond. • Ligase “Joins” pieces of DNA by making a 3`- 5` phosphodiester bond Topoisomerases: Change state of DNA supercoiling [demonstrate with model] [Fig.27-2] Topoisomerases: Change state of DNA supercoiling Add topoisomerase 0 min 5 min 30 min 2 kinds of supercoils Negative (right-handed) Positive (left-handed) Topoisomerases can convert (+) to (-) supercoils Topoisomerase(s) II 2 strands cut Right-handed supercoils (DNA gyrase uses ATP) Topoisomerase(s) I 1 strand cut Left-handed supercoils [Helicase in DNA synthesis makes NO cuts, uses ATP] Topoisomerase I – cuts one strand Negative (- 5) supercoils Positive (- 4) cut Topoisomerases II make 2 cuts (Ex: DNA Gyrase) Cuts 2 strands (-) 1 Supercoil changes (+) 1 Mechanism of DNA Gyrase (a Topoisomerase II) 5`-P linked to Tyrosine on A subunits +1 Left-handed [“Bad” (Stress)] -1 Net –2 linking # Right-handed [“Good”] DNA Ligase: Makes a 3`- 5` phosphodiester bond • • requires a “nick”(break) in a 3’-5’ phosphodiester bond. “Joins” pieces of DNA by making a 3`- 5` phosphodiester bond l l l l l l l l l l l .. 3`OH 5`P Nucleophilic attack l l l l l l l l l l l l l l l l l l l l l l DNA nick -P- l l l l l l l l Joins a 3`OH with a free 5`-Phosphate of Adjacent bases DNA LIGASE Reaction 2Pi Å PPi New 3`- 5` phosphodiester bond AMP Summary: DNA TOPOISOMERASES DNA LIGASE uses ATP Makes new 3`- 5` bond TOPOISOMERASE I Adds (+) supercoils No ATP required 1-strand cut HELICASE Adds (+) supercoils Needs ATP No Cuts DNA GYRASE Adds (–) supercoils 2 strand cut Needs ATP CHAPTER 27: DNA STRUCTURE, REPLICATION AND REPAIR TOPIC REVIEW 1) DNA STRUCTURE Æ Models vs X-ray Æ Static vs Dynamic 2) DNA-PROTEIN INTERACTIONS Æ Sequence-specific vs non-specific 3) DNA TOPOISOMERASES – Changes in state of DNA Æ Cutting and sealing strands CHAPTER 27: DNA STRUCTURE, REPLICATION AND REPAIR LECTURE TOPICS 4) DNA REPLICATION Æ E. coli chromosome Æ The players and the process 5) DNA RECOMBINATION 6) DNA MUTATIONS AND REPAIR DNA REPLICATION • DNA POLYMERASES • THE REPLICATION PROCESS DNA polymerase I (Pol I) has 3 different activities 1. Template- Directed DNA polymerase (5`Æ 3` Polymerase) [Processive enzyme adds 20 bases at 10/sec] 2. Proofreading: 3`Æ 5` Exonuxlease (corrects last error) 3. Error Correcting 5`Æ3` exonuclease (repairs old errors) DNA polymerase I (Pol I) reaction mechanism: 5’ to 3’ polymerase [see Ch.5 notes] 5’ Nucleophilic attack 3` New base 5’ ** Error Rate: 1/10,000 bases (10-4) Pol I proof reading exonuclease (3’ Æ 5’ editing) • removes wrong base if inserted (leaves a 3`OH) Error rate is also 1x10-4 Total Error Rate for Pol I DNA synthesis and editing = 10-4 x 10-4 = 10-8 5’ 3’ The “Central Dogma” of molecular biology 10 -4,-5 10-3, -4 10-8 Transcription translation DNA RNA Replication Reverse transcription DNA virus Retrovirus RNA Virus PROTEIN Prions 10-4 FEATURES OF PROCESSES Accuracy, Signals, Stage 8 One error in 10 bases polymerized 6 7 In E. coli, 4x10 bases x 2 DNA strands ~ 10 bases per replication This is 1 mistake in 10 cells Pol I exonuclease (5’ Æ 3’ editing) removes pre-existing errors mismatches (Exonuclease) 5` 5` cut 3` Pol I Klenow fragment 5`Æ3` 5`Æ3` Question: Is Pol I sequence specific?? + 2- Mg2 metal ions in Pol I active site play a role in 5’ to 3’ polymerase mechanism d 3` OH α-P Pol I (donor) H-bonds to base pair acceptors T A * Minor Groove H-bonds * Base pair functional group acceptors* are same for A-T and G-C base pairs Pol I : Incoming dNTP causes formation of tight binding pocket in 5’ to 3’ polymerase d d Pol I 3`Æ 5` exonuclease (edits a mistake) Move cut strand to exonuclease site Cut wrong base Leave 3`-OH Unzip base-paired section Observation : E. coli mutants lacking Pol I replicate DNA and grow normally. How?? DNA POLYMERASES II and III discovered (late 1960’s) Æ Have 5` to 3` polymerase (like Pol I) and proofreading 3` to 5` exonuclease Æ No 5` to 3` exonuclease activity ÆPol III used for chromosomal DNA replication (processive – 1000 base pairs / second) Æ Many other proteins also involved in replication DNA POLYMERASE III (Pol III) Catalysis Holoenzyme is an asymmetric dimer Pol III is processive : Pol III β2 - dimers ÆAdds thousands of bases Æ1000 / sec (Pol I is 10 / sec)* * Pol III is 100 times as fast as Pol I Question: How many minutes to replicate E. coli DNA? DNA POLYMERASES: SUMMARY DNA POLYMERASE I – Three different activities • • • • Template directed 5`Æ3` polymerase Proofreading (3`Æ5' exonuclease) Error-Correcting (5`Æ3` Exonuclease) E. coli mutants lacking Pol I have normal growth and DNA replication DNA POLYMERASES II AND III • Have 5` to 3` polymerase and proofreading 3` to 5` exonuclease • • Pol III replicates the E. coli chromosome Many other proteins are also involved Ori C : 254 b.p. Origin of Replication [Start signal for Initiation of replication] E. Coli chromosome replicating looks like this: (theta structure) Replication fork Replication fork ELONGATION: Direction of DNA synthesis is 5`Æ 3` Apparent 3` Æ 5` (Discuss first) Actual 5` Æ 3` (as always) (Discuss later) Helicase unwinds DNA • Uses ATP as energy • Introduces positive supercoils Initiation of DNA synthesis (An RNA primer is extended 5’ – 3’) (An RNA polymerase) • Both strands • Almost all chromosome DNA synthesis Termination of DNA synthesis Pol I 5`Æ3` Exonuclease Pol I removes primer Pol I 5`Æ3`synthesis DNA ligase Okazaki fragments joined Some DNA replication proteins in E. coli (+/-) supercoils added •E. Coli chromosome contains 400,000 turns of helix •Need 100 turns / second E. Coli replication fork (+) (SSB) (-) has gyrase too! 5’ Pol III dimer holoenzyme synthesizes both strands at fork. Inverted loop Primer Lagging strand (1,000 bases average length) Leading strand Eukaryotic chromosome replication Elongation is bi-directional from thousands of forks. Ex : Drosophila chromosome (size – 62 x 106 bp) Replication rate is 2.6 kb/min/origin. To replicate the chromosome: 16 days with only one origin Actual rate : < 3 minutes Need > 6000 replication forks!! Eukaryotic chromosome: Problem at end of replication (telomere) [New histones] [Old histones] ?? One daughter molecule would get shorter and shorter! End of Chromosome termination solution TELOMERASE • • • • A ribonucleoprotein complex (RNA + protein) A Reverse transcriptase with an RNA template Processive Adds 100’s of short repeated sequence to incomplete 3` ends of chromosomes Telomere 100,s of GGGTTG added RNA Primer New DNA Telomerase Many repeats of new DNA TELOMERASE Chromosome 3` end The end of the telomere (May 1999) The new view Telomere: Repeated sequences form base pairs 5’ 3’ CHAPTER 27: DNA STRUCTURE, REPLICATION AND REPAIR LECTURE TOPIC 5) DNA RECOMBINATION DNA Recombination: Occurs between molecules that have similar sequences Homologous Recombination results in: • Gene replacement • Gene disruption * * * * * [Shared sequences] Recombined Gene with some different bases [Fig.6-31] “Recombinase” (Cre- a Type I topoisomerase) * * * * * * * * CHAPTER 27: DNA STRUCTURE, REPLICATION AND REPAIR LECTURE TOPIC 6) DNA MUTATIONS AND DNA REPAIR 4 SIGNS of MALIGNANT MELANOMA MUTATIONS ARISE FROM MISMATCHED BASES IN DNA • Persistent replication errors are actually only 10-9 to 10-10 [DNA repair improves error from 10-8] • • Chemical mutagens Ultraviolet light (Sunlight) DNA REPAIR • • • Base excision [uracil removal] T-T dimer removal [defect in Xeroderma pigmentosum] Mismatch repair [defects in colorectal, stomach, uterine cancers] IS A MUTAGENIC AGENT ALSO CARCINOGENIC?? • Ames test [Reversion of Salmonella His- to His+ phenotype] A replication error C A * C:A mismatchÆ mutation A:T to G:C A transition mutation [purine to purine] * Chemical Mutagen : Nitrous acid (HNO2) Deamination causes A:T to G:C transitions A “A” C C C:A mismatchÆ mutation * HNO2 also deaminates C to U: causes G:C to A:T transitions Base Analog Mismatch: Thymine analog 5-BU 5-BU:T mismatchÆ A to T mutation “T” Looks like C G Should be A Intercalating mutagens fit between adjacent base pairs • cause base insertions, leading to translational frameshifts Same size as a base pair DNA Chemical Adducts Epoxide [reacts withN7 of Guanine and forms covalent link] DNA Repair: 3 types Altered base (3-CH3-Adenine) * * 1) Base excision repair * * 2) In place repair (pyrimidine dimers) Repair of T-T dimers Remove several bases 3) Excision repair T-T dimers (adjacent bases on same strand of DNA) * Sunlight: UV light causes T-T dimer formation * Repair of T-T dimers Cut T-T Cut excinuclease OH 5` P Pol I 3` Pol I Ligase 5` 3` C4- (NH2) to C4- (C=O) C U * * * Remove uracil T Cut 3`-5` Phosphodiester bond Repair of uracil in DNA: Pol I + Ligase [uracil would lead to C to T transition] Mismatch Repair: Occurs soon after a DNA replication error Template New DNA No CH3- A on new DNA Exonuclease Endonuclease Up to 2000 bases removed Pol III Synthesize again Ligase Triplet repeat expansions in eukaryotic DNA: (Associated with neurological diseases) Loop lets red strand get longer with 3 more repeats added Ames test: Are mutagens also carcinogens? Medium lacks histidine His- mutants Mutagen + liver extract His+ revertants CHAPTER 27: DNA STRUCTURE, REPLICATION AND REPAIR SEE KEY CONCEPTS: P.1 ONLINE LECTURE NOTES