Chapter28Powerpoint 11.45.23 PM

Chapter28Powerpoint 11.45.23 PM - CHAPTER 28: DNA...

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Unformatted text preview: CHAPTER 28: DNA STRUCTURE,REPLICATION, REPAIR LECTURE TOPICS 1) DNA STRUCTURE Models vs X-ray Static vs Dynamic 2) DNA-PROTEIN INTERACTIONS Sequence-specific vs non-specific 3) DNA TOPOISOMERASES – Changes in state of DNA Cutting and sealing strands 4) DNA REPLICATION E. coli chromosome The players and the process 5) DNA RECOMBINATION 6) DNA MUTATIONS AND REPAIR Francis Crick James Watson 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 Base roll causes bending DNA: Bases are not in a plane N9 N1 PROPELLER TWISTING B-DNA A-DNA (and RNA) 2’ up 2’ down RNA 2`OH Steric Hindrance Won’t fit B-DNA helix 3` 5` phosphodiester bond Z – DNA: exists but Function unknown Z DNA B DNA-RNA or (RNA-RNA) A DNA-RNA RNA-RNA Most DNA RARE Table 27-1 frp,, 5th CHAPTER 28: DNA STRUCTURE,REPLICATION, REPAIR LECTURE TOPICS 1) DNA STRUCTURE Models vs X-ray Static vs Dynamic 2) DNA-PROTEIN INTERACTIONS Sequence-specific vs non-specific 3) DNA TOPOISOMERASES – Changes in state of DNA Cutting and sealing strands 4) DNA REPLICATION E. coli chromosome The players and the process 5) DNA RECOMBINATION 6) DNA MUTATIONS AND REPAIR 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 * * * = CH 3 in host E.coli !! Asymmetrical DNA Recognition Site!! [Fig.9-37; pp. 259-266 in text for Eco RV discussion] 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 [Distortion of 5’-G-A-3’ facilitates Mg-binding and catalysis] [Fig.9-40] 250 2 50 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) EcoRV β-turn loops H-bond with DNA Specific H-bonds in each EcoRV monomer * * * ** [Fig.9-39] ** * * * * CH3(A) blocks this H-bond Evolution: DNA sequence elements are conserved in active sites of some Type II restriction enzymes EcoRI recognition site GAATTC • CTTAAG l l lll l Each DNA strand forms 6 H-bonds with Glu and Arg residues of Eco RI. • • 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 CHAPTER 28: DNA STRUCTURE, REPLICATION, REPAIR LECTURE TOPICS 1) DNA STRUCTURE Models vs X-ray Static vs Dynamic 2) DNA-PROTEIN INTERACTIONS Sequence-specific vs non-specific 3) DNA TOPOISOMERASES – Changes in state of DNA Cutting and sealing strands 4) DNA REPLICATION E. coli chromosome The players and the process 5) DNA RECOMBINATION 6) DNA MUTATIONS AND REPAIR 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 (state of supercoiling). • 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 • Cut and seal 3’-5’ phosphodiester bonds in DNA [demonstrate with model] [Fig.27-2] 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 lllllllllll .. 3`OH 5`P Nucleophilic attack lllllllllllll lllllllll 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 PPi New 3`- 5` phosphodiester bond AMP Topoisomerases: Change state of DNA supercoiling + topoisomerase 0 min 5 min 30 min 2 kinds of supercoils (-) Negative (right-handed) (+) Positive (left-handed) 2 kinds of supercoils (+) Positive (left-handed) (-) Negative (right-handed) Topoisomerases can convert (+) to (-) supercoils Topoisomerase(s) II 2 strands cut add (-) Right-handed supercoils (DNA gyrase, in DNA synthesis, uses ATP) Topoisomerase(s) I 1 strand cut add (+) Left-handed supercoils [But Helicase in DNA synthesis makes NO cuts, uses ATP] Topoisomerase I – cuts one strand Adds (+) supercoils Negative (- 5) Positive (- 4) covalent link to tyrosine reseal cut pass 1 strand through Topoisomerases II make 2 cuts (Ex: DNA Gyrase) Cuts 2 strands (-) 1 Supercoil changes (+) 1 Mechanism of DNA Gyrase (a Topoisomerase II) 2 (+1) Net –2 linking # Left-handed [“Bad” (Stress)] 2 2 (-1) Right-handed [“Good” (less stress)] 5`-P linked to Tyrosine on A subunits Topoisomerases II (DNA gyrase) Inhibitors: quinolones Block breaking and joining of DNA chains Used for urinary tract, other infections Anthrax (Cipro) *** *** [gyrase] Anthracyclene Topoisomerase II Inhibitors: “Intercalate” between base pairs 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 (A TOPOISOMERASE II) Adds (–) supercoils 2 strand cut Needs ATP CHAPTER 28: DNA STRUCTURE, REPLICATION, REPAIR LECTURE TOPICS 1) DNA STRUCTURE Models vs X-ray Static vs Dynamic 2) DNA-PROTEIN INTERACTIONS Sequence-specific vs non-specific 3) DNA TOPOISOMERASES – Changes in state of DNA Cutting and sealing strands 4) DNA REPLICATION E. coli chromosome The players and the process 5) DNA RECOMBINATION 6) DNA MUTATIONS AND REPAIR October 28, 2007 Arthur Kornberg, 89; the Stanford University Nobel laureate died of respiratory failure Friday at Stanford Hospital. Dr. Kornberg was the first to synthesize DNA in a test tube (1957). His identification and characterization of the enzymes used by cells to manufacture DNA laid the foundation for the biotechnology industry. [By Thomas H. Maugh II, Los Angeles Times Staff Writer] Arthur Kornberg discovered all of this: 1) All enzymes that make the A,G,C,U, and T nucleotides for DNA and RNA 2) The nucleotide salvage pathways 3) 1957- DNA polymerase I [papers rejected in 1957, accepted in 1958. He called the enzyme product “DNA” but reviewers wanted proof that they carried genes!] 4) 1959 Nobel prize in Medicine for synthesis of DNA in a test tube. 5) DNA ligase (1967, along with 3 other groups) 6) 1967 – synthesized infectious phage dsDNA in a test tube. [Same day as published, Lyndon Johnson called it “life in a test tube” – embarrassing!] 7) Repair function of DNA Polymerase I 8) Arthur and son Thomas discovered DNA Pol II and III 9) Showed that folic acid was an essential nutrient 10) Showed that sulfa drugs looked like a precursor to folic acid and that they prevented vitamin K formation in bacteria 11) Son Roger won 2006 Nobel prize in Chemistry DNA REPLICATION • DNA POLYMERASES DNA polymerase I (Pol I) has 3 different activities 1. Template- Directed DNA polymerase (5` 3` Polymerase) [A Processive enzyme: adds 20 bases at 10 bases/sec] 2. Proofreading: 3` 5` Exonuxlease (corrects last error) 3. Error Correcting 5` 3` exonuclease (repairs old errors) DNA polymerase I (Pol I) reaction mechanism: 5’ to 3’ polymerase [see Chapter 4 notes] 5’ Nucleophilic attack 3` .. New base 5’ ** Error Rate: 1/10,000 bases (10-4) Pol I proof reading exonuclease (3’ 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 = 1/104 x 1/104 = 1/108 5’ 3’ Pol I 3` 5` exonuclease (proofreading - edits a mistake) Move strand to exonuclease site Cut wrong base Leave 3`-OH Unzip base-paired section Try again with 5’-3’ polymerase activity The “Central Dogma” of molecular biology 10 -4,-5 10-3, -4 10-8 Transcription translation DNA RNA Replication Reverse transcription DNA virus Retrovirus PROTEIN RNA Virus Prions 10-4 FEATURES OF PROCESSES • Accuracy, Signals, Stages • One error in 108 bases polymerized (DNA) • In E. coli, 4.6x106 bases x 2 DNA strands( ~ 107 bases/replication) • This is only 1 DNA replication mistake in 10 cells!! Pol I exonuclease (5’ removes pre-existing errors 3’ editing) mismatches (Exonuclease) 5` 3` 5` cut • 5’ to 3’ polymerase • 5’ to 3’ “nick translation” • (a DNA repair function) 3` Pol I Klenow fragment (no 5’-3’ exonuclease domain) 5` 3` 5` 3` Question: Is Pol I sequence specific?? ++ 2 metal (Mg2 ) 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 Accuracy determined by base “shape complementarity” [analogs that fit will direct correct pairing] Pol I : Incoming dNTP causes formation of tight binding pocket in 5’ to 3’ polymerase d d Pol I is “correct base –pair” (not sequence) specific Base “shape selection” chooses correct Watson-Crick pairs Observation : E. coli mutants lacking Pol I replicate DNA and grow normally. How?? DNA POLYMERASES II and III discovered (late 1960’s) • Have 5` to 3` polymerase (like Pol I) and proofreading 3` to 5` exonuclease • No 5` to 3` exonuclease activity •Pol III is used for chromosomal DNA replication (processive – 1000 base pairs/second) • Many other proteins also involved in replication • Pol II biological function not well understood. DNA POLYMERASE III (Pol III) DNA Processivity Catalysis (5’ to 3’) Proofreading (3’ to 5’) Holoenzyme is a huge asymmetric dimer Pol III is processive : Pol III β2 - dimer Adds thousands of bases 1000/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` 3` polymerase Proofreading (3` 5` exonuclease) Error-Correcting (5` 3` 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 DNA REPLICATION THE REPLICATION PROCESS E. Coli chromosome during replication looks like this: (theta structure) Replication fork Origin of replication (OriC) Replication fork end Ori C : *245 b.p. Origin of Replication [Start signal for Initiation of replication] Dna A protein assembles with Ori C [replication start complex; favors A-T rich strand separation] Helicase (Dna B protein): unwinds DNA during replication • Uses ATP as energy • Introduces positive supercoils • Helicases are evolutionarily conserved [Fig. 28-25] Singe-stranded DNA Initiation of DNA synthesis (An RNA primer is extended 5’ – 3’) 1) (An RNA polymerase) • Both strands 2) • synthesizes almost whole chromosome ELONGATION: Direction of DNA synthesis is 5` 3` Appears to be growing 3` 5` Actual is 5` 3` (as always) Pol I 5` 3` Exonuclease Pol I 5` 3`synthesis fills gap Pol I removes primer DNA ligase Some DNA replication proteins in E. coli (+) supercoils added and removed (-) • E. Coli chromosome contains 400,000 turns of helix • Need 100 turns/second E. coli replication fork (SSB) (+) (-) gyrase! 5’ E. coli replication fork (-) Gyrase (+) III Pol III holoenzyme at the E. coli replication fork Processivity Catalysis (5’ to 3’) Proof-reading (3’ to 5’) Pol III holoenzyme at the E. coli replication fork loop inverts polarity 5’ to 3’ Asymmetric dimer [reversed relative to DNA polarity] 5’ to 3’ Pol III dimer holoenzyme synthesizes both strands at fork. Inverted loop Primer [5’ to 3’] Lagging strand (1,000 bp average length) [5’ to 3’] Leading strand DNA REPLICATION THE REPLICATION PROCESS http://highered.mcgraw-hill.com/olc/dl/120076/micro04.swf http://highered.mcgraw-hill.com/olc/dl/120076/bio23.swf CHAPTER 28: DNA STRUCTURE, REPLICATION, REPAIR LECTURE TOPICS 4) DNA REPLICATION - Eukayotes 5) DNA RECOMBINATION 6) DNA MUTATIONS AND REPAIR Eukaryotic chromosome replication Elongation is bi-directional from thousands of forks. Replication bubbles are 30-300 kb apart. Ex : A Drosophila chromosome (size – 62 x 106 bp) Replication rate is 2.6 kb/min/origin. To replicate the chromosome will take 16 days with only one origin Actual rate is < 3 minutes: Need > 6000 replication forks!! Eukaryotic chromosome: Problem at end of replication (telomere) [New and old histones are distributed among leading and lagging strands] ?? One daughter molecule would get shorter and shorter! Telomerase adds to 3’-end Solution to maintain end of chromosomes TELOMERASE • • • • A ribonucleoprotein complex (RNA + protein) A Reverse transcriptase with an RNA template Processive activity Adds 100’s of short repeated sequence to 3` ends (SS) of chromosomes Telomere 100’s of GGGTTG added to 3’-end Telomerase mechanism RNA template base pairing ** New DNA Template shifts ** New DNA Many repeats of new DNA TELOMERASE Chromosome 3` end Telomerase Animation: http://www.biochem.arizona.edu/classes/bioc462/462bh2008/462bhonorsprojects/462bhonors1999/bentley/telomeraction.html The very end of the telomere (May 1999) Lasso structures: Repeated sequences form base pairs [3’- end completed by telomerase 5’ 3’ [5’- end completed by polymerase CHAPTER 28: 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.5-34] “Cre Recombinase” ( a Type I topoisomerase) * * * * Cut ends are 5’ -OH * * * * CHAPTER 28: 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 [Pol I and other DNA repair improves error from 10-8] • • Chemical mutagens Ultraviolet light (Sunlight) DNA REPAIR MECHANISMS • • • 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] Ames test: Are mutagens also carcinogens? Medium lacks histidine x + Mutagen (x) + liver extract + 109 Salmonella His- cells His+ revertants Mutations cause replication errors C * A C:A mismatch mutation A:T to G:C mismatch A transition mutation [purine to purine] * Base Analog Mismatch: Thymine analog 5-BU 5-BU:G mismatch A to G mutation (A:T to G:C) “T” Looks like C G Should be A Intercalating mutagens: lead to translational frameshifts • fit between base pairs [like anthracyclene gyrase inhibitors] • cause base insertions or deletions (DNA polymerase gets confused) Same size as a base pair DNA Chemical Adducts Epoxide • Epoxide reacts with N7 of Guanine, forming a covalent link. • Leads to replication errors G Chemical Mutagen : Nitrous acid (HNO2) Deamination of A causes A:T G:C transitions A “A” C:A mismatch * HNO2 also deaminates C to U (looks like T): - causes G:C to A:T transitions - But C to U can be repaired CC mutation Types of DNA Repair: 3 examples Altered base (Ex.: 3-CH3-Adenine) * * 1) Base excision repair * * In place repair of pyrimidine dimers 2) Repair of T-T dimers Remove several bases 3) Excision repair C4- (NH2) to C4- (C=O) C U * Remove uracil * [HNO2] * A:T Cut 3`-5` Phosphodiester bond Pol I + Ligase Repair of uracil in DNA: [uracil would lead to C to T transition] 1) Base excision repair Remove base AP is: apurinic (no A or G) or apyrimidinic (no C or T) Pol I + Ligase Cut 3`- 5` phosphodiester bond T-T dimers (adjacent bases on same strand of DNA) T-T * Sunlight: UV light causes T-T dimer formation * Repair of T-T dimers Cut T-T Cut excinuclease 3’- OH 5` 5’-P Pol I 3` Pol I Ligase 5` 3` Mismatch Repair: Occurs soon after a DNA replication error Template New DNA * [CH3-A on old DNA] No CH3-A on new DNA ** Exonuclease Endonuclease Up to 2000 bases removed Pol III Synthesize again with Pol III Ligase Triplet repeat expansions in eukaryotic DNA • Associated with neurological diseases • [CAG] repeats Poly-Gln associated with Huntington’s disease] 1) New DNA base pairs break 2) DNA forms new base pairs leaving a ssDNA loop 3) SS-DNA loop lets red strand get longer with 3 more repeats added Triplet repeats CHAPTER 28: DNA STRUCTURE, REPLICATION AND REPAIR SUMMARY of KEY CONCEPTS: • DNA structure is dynamic. It can be bent, kinked, unwound, and have different helical structures. • Proteins interact with DNA in all biological activities involving DNA. • DNA must be unwound to replicate. • Topoisomerases catalyze changes in supercoiled state of DNA. • DNA replication has three distinct phases (initiation, elongation, and termination). • Termination is different at telomeres of eukryotic chromosomes. • DNA replication is very accurate (1x10-8 mistakes/base). CHAPTER 28: DNA STRUCTURE, REPLICATION AND REPAIR SUMMARY of KEY CONCEPTS: • DNA molecules can “recombine” if they have similar sequences. • Mutations have several causes and involve base sequence changes. • DNA repair corrects errors using highly evolved correction systems. ...
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