Class Note for BIOC 461 at UA
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Date Created: 02/06/15
CHAPTER 27 DNA STRUCTURE REPLICATION REPAIR LECTURE TOPICS 1 DNA STRUCTURE 9 Models vs Xray 9 Static vs Dynamic 2 DNAPROTEIN INTERACTIONS 9 Sequencespecific vs nonspecific 3 DNA TOPOISOMERASES Changes in state of DNA 9 Cutting and sealing strands 4 DNA REPLICATION 9 E coli chromosome 9 The players and the process 5 DNA RECOMBINATION 6 DNA MUTATIONS AND REPAIR Ecoli is Mr DNA replication Mr Transcription Mr Translation Mr Control of gene expression The Boss actually it39s Edward The E is Escherichia but you can call me Ed Models vs Real DNA structure from xray diffraction WatsonCrick Model Real BDNA structure 36 turnl base pair 2842 turnbase 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 sequence specific dependent sequence provides unique 3D fit for proteinDNA interactions REAL BDNA from Xray structure Major gvoove Major gmove BENDING OF DNA B DNA Bases are not in a plane K Propeller twist PROPELLER TWISTING REAL BDNA from Xray structure Show Movie CHAPTER 27 DNA STRUCTURE REPLICATION REPAIR LECTURE TOPICS 1 DNA STRUCTURE 9 Models vs Xray 9 Static vs Dynamic 2 DNAPROTEIN INTERACTIONS 9 Sequencespecific vs nonspecific 3 DNA TOPOISOMERASES Changes in state of DNA 9 Cutting and sealing strands 4 DNA REPLICATION 9 E coli chromosome 9 The players and the process 5 DNA RECOMBINATION 6 DNA MUTATIONS AND REPAIR Base pairs Hbonding properties Maj or g mmve Major 9 move Minorgroove Minorglroove Adenine Thyrnirne Guanine Cytasin AT G C Bases are Hdonors D or acceptors A ADNA and RNA DNARNA or RNARNA Z DNA exists but Function unknown Z B A Most DNA 739 DNARNA P r RNARNA 1 TABLE 211 Comparison of A B3 and EDNA Helix type A B Z tilqu Erna dest lnterm ediate Narrowest Rim per base pair 33 A 3 Helix diameter 255 A 23 rill 391 SAP A gtE rrmv sense Right handed Rig lit handed handed 39Cilymsidic hand anti anti alternating anti and syn Base airs 21 turn Hf helix ll 12 gt Pitch per turn ll helix 253 354 45f gtPl ilt 1f base pairs fmm 391 939 narmal m helix axis rlajmr grriove Narrow and little and F at veryquot deep quite deep Very hrth Narmw and Very narrow and shajlluw quite deep and d p Nonspecific DNA sequence independent Specific DNA Sequence mattersll Nonspecific interactions Deoxyribonuclease I Arg I Lys have charge in protein EcoRV restriction enzyme recognition site I Sequencespecific interactions 2fold symmetry Symmetry axis Asymmetrical DNA Recognition Site Fig937 DNA bases form specific Hbonds with loop of EcoRV protein pturn Sequence specific Interactions in DNA major groove EcoRV bends kinks DNA by 50 1 Fig940 EcoRVptum loops Specific Hbonds in each Hbond with DNA EcoRV monomer Fig939 Evolution DNA sequence elements are conserved in active sites of some Type II restriction enzymes EcoRl recognition site t GAATTC I I I I I CTT G Each DNA strand forms 6 Hbonds with Glu and Arg residues of Eco RI the enzyme A total of 12 Hbonds form in EnzymeDNA complex EcoRl DNA complex One side 7 e A AwT T C c r wTKA A G39 39 Half a helix turn Top Two kinds of EcoRlDNA Interactions dipolephosphate backbone interactions at a specific location Meg a Hem Protein CJ e specific Hbonds G base we C Nquot 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 39 Ligase requires a nick break in a 3 5 phosphodiester bond 39 Ligase Joins pieces of DNA by making a 3 5 phosphodiester bond Topoisomerases Change state of DNA supercoiling Unwound Overwound demonstrate with model Fig272 TopOIsomerase Change state of DNA superco ng Add topoiso erase K C A B Relaxed DNA 9 F Highly supercoiled DNA 0 min 5 min 30 min 2 kinds ofsupercoils Relaxed DNA Lk 20 ALk 2 AL 2 Positive Negative x I lefthanded righthanded Negative Positive supercoil supercoil Lk18 Lk22 Topoisomerases can convert to supercoils Positive Negative supercoil supercoi 74k 18 L1 22 Topoisomerases ll Topoisomerases l 2 strands cut 1 strand cut Right handed supercoils Left handed supercoils Helicase in DNA synthesis makes NO cuts uses ATP DNA gyrase uses ATP Topoisomerase cuts one strand supercoils Negative 5 Positive 4 r hfha f w wi p lxx lw lw lxw lmv lxw h hp g hwdlwqw lxw thy Negatively supercoiled DNA Topoisomerases make 2 cuts Ex DNA Gyrase I 3 segment T segment Mechanism of DNA Gyrase a Topoisomerase ll DNA unlah ng 5 P linked to Tyrosine on A subunits Lefthanded Bad Stress m w H H10 cunmmng 39 ALE Right anded Good I DNA Ligase Makes a 3 5 phosphodiester bond I requires a nick break in a 3 5 phosphodiester bond Joins pieces of DNA by making a 3 5 phosphodiester bond 5 P DNAnick W Nucleophilic attack Adjacent bases Joins a 3 OH with a free 5 Phosphate of l DNA LIGASE Reaction 0 DNA strand 3 O T O 5 DNA strand iDNh strand 3 0 DNA Ligase ATP MAW or 139 39 W O H o P o 539 DNA strand O 1 New 3 5 phosphodiester bond Summary DNA TOPOISOMERASES DNA LIGASE uses ATP Makes new 3 5 bond TOPOISOMERASE l Adds supercoils No ATP required 1strand 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 9 Models vs Xray 9 Static vs Dynamic 2 DNAPROTEIN INTERACTIONS 9 Sequencespecific vs nonspecific 3 DNA TOPOISOMERASES Changes in state of DNA 9 Cutting and sealing strands CHAPTER 27 DNA STRUCTURE REPLICATION AND REPAIR LECTURE TOPICS 4 DNA REPLICATION 9 E coli chromosome 9 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 9 3 Polymerase Processive enzyme adds 20 bases at 10lsec 2 Proofreading 3 9 5 Exonuxlease corrects last error 3 Error Correcting 5 93 exonuclease repairs old errors DNA polymerase I Pol I reaction mechanism 5 to 3 polymerase see Ch5 notes 5 3 Primer strand 3 aner strand D 13 Nucleophilic g g X attack H10 3 a 6 2 o o 9 l a I 0 gr 0 3r a m m quotrf quot I x EL We rF Oj New base a o a 04quot 0 0 t a a D E m 5 HD 5 H0 5 aw Error Rate 11oooo bases 10quot Pol proof reading exonuclease 3 9 5 editing removes wrong base if inserted T T ETA Hydmlysia site 3 3 5 exonucleaSe activity leaves a 3 OH T 3H0 Error rate is also 1x104 Total Error Rate for Pol DNA synthesis and editing 104 x 104 108 2 5 T 3 The Central Dogma of molecular biology I Trancri tion translation CDNA p RNA 3 gt PROTEIN Replication Meg Q transc ption DNA virus Retrovirus RNA Virus Prions FEATURES OF PROCESSES CI Accuracy Signals Stage El One error in 108 bases polymerized El In E coli 4x106 bases x 2 DNA strands 107 bases per replication El This is 1 mistake in 10 cells Pol exonuclease 5 9 3 editing removes preexisting errors mismatches Hyd rolysis site 539 gt 339 nudeage activity I A Exonuclease WW 5 D 5 cut Pol Klenow fragment 5 93 Polymerase i 3 domain I 1quotf39 Q 4 3 m 4 d S t e f n h h wu li Exgnuc ease gt v L dumain 20A site Question Is Pol sequence specific 2 M92 metal ions in Pol active site play a role in 5 to 3 polymerase mechanism Pol donor Hbonds to base pair acceptors Base pair functional group acceptors are same for AT and GC base pairs Pol Incoming dNTP causes formation of tight binding pocket in 5 to 3 polymerase Pol 3 9 5 exonuclease edits a mistake Move cut strand to exonuclease site Migration to Template exunudease Site strand Exunuclease acme me Leave 3 OH Unzip basepaired section Observation E coli mutants lacking Pol l replicate DNA and grow normally How DNA POLYMERASES II and Iquot discovered late 1960 s 9 Have 5 to 3 polymerase like Pol I and proofreading 3 to 5 exonuclease 9 No 5 to 3 exonuclease activity 9Pol lll used for chromosomal DNA replication processive 1000 base pairs I second 9 Many other proteins also involved in replication DNA POLYMERASE Pol III Catalysis Holoenzyme is an asymmetric dimer Pol III is processive 9Adds thousands of bases 91000lsec Pol is 10 I sec Pol III is 100 times as fast as Pol Question How many minutes to replicate E coli DNA DNA POLYMERASES SUMMARY DNA POLYMERASE Three different activities Template directed 5 93 polymerase Proofreading 3 9539 exonuclease ErrorCorrecting 5 93 Exonuclease E coli mutants lacking Pol l have normal growth and DNA replication DNA POLYMERASES H AND Iquot Have 5 to 3 polymerase and proofreading 3 to 5 exonuclease Pol III replicates the E coli chromosome Many other proteins are also involved L m Tandem array of I S rner sequences AT rich w CT 5 3quot gt4 CTNTTNTTTTJS39 CANAANAAAA7539 nsensus sequence 9 Ori C 254 bp Origin of Replication Start signal for Initiation of replication E Coli chromosome of 9M replicating looks 1 M like this 39 gv theta structure Replication fork 9 M D O ELONGATION Direction of DNA synthesis is 5 9 3 Replication Parental fork 5 DNA39 I lt 9 3 Apparent 3 9 5 Leading strand V 3 5 Discuss first 3 5 NW Actual 5 9 as always 5 Lagging strand Discuss later Parental DNA Helicase unwinds DNA Uses ATP as energy Introduces positive supercoils In tion of DNA synthesis An RNA primer is extended 5 3 DNA template 339 5 iPrimaseAn RNA polymerase 339 5 5 3 RNA Primer Both strands DNA Eoxrnerase ll Almost all chromosome 3 5 DNA synthesis 5 3 New DNA Termination of DNA synthesis Template strand 539n L DNA k RNA primer KM DNA fragment fragment Hemowl of FINA pnmer Pol 5 93 Exonuclease and gapfiilinnbv Pol l removes primer DNA polymerase I 3i N DNA aw i I Pol 5 93 syntheSIs 339laii II 5 3 DNA ligase Okazaki fragments joined 39 6 l a l Comnleted daughter strand Some DNA replication proteins in E coli r Size Mo Protein Role Md pefZL lfs e W the double helix 300 E u protein Primase SW so v 888 Stabilizes single stranded regions 74 300 9 39W 400 250 sxnthw gs DNA 900 39 39 holoenzyme DNA polymerase I Erases primer and fiils gaps 103 DNA Iigase Joins the ends of DNA 74 300 supercoils added E Coli chromosome contains 400000 turns of helix Need 100 turns second E Coli replication fork Helkase ssB Singlemandch binding protein N has gyrase too Plimosome Pol III dimer holoenzyme 39 Inverted loop synthesizes both strands at fork Primer Lagging strand 1000 bases average length Leading strand Eukaryotic chromosome replication Mn W lt gt Elongation is bidirectional from thousands of forks Ex Drosophila chromosome size 62 x106 bp Replication rate is 26 kbminorigin To replicate the chromosome 16 days with only one origin Actual rate lt 3 minutes Need gt 6000 replication forksll I Eukaryotic chromosome Problem at end of replication I DNA 31quot J1 Replication Leading strand Old histones mu 3 339m 5r telomere New histones Lagging strand HNA primer Erasureuf one prlmer daughter Incomplete 5quotquot 3 mOIGCUIG daught r arm 5 would get shorter Complet emu 339 and daughter awn 5r shorter End of Chromosome termination solution TELOMERASE 39 A ribonucleoprotein complex RNA protein 39 A Reverse transcriptase with an RNA template 39 Processive 39 Adds 100 s of short repeated sequence to incomplete 3 ends of chromosomes Telomere 4 1005 of GGGTTG added Ielnvmere r 3 OH 3quot HIT 4 21211 5 A cccug RNA Prlmer 39lfvalomera e RNA 39539 539 Elongation TD x New DNA my ACCCCAA I DH 339 OH 3 13 GGGT39IGCGG39 TI 5quot AA fa C CCCAA L s ub z Elongation l or FGGGTTGGGGTT EE1iITGOH 2 539 Ac LAA I k 9 39lmrvslocaaiun l I Many repeats CGG1IGGGHGGGGTTG AUH 3 f DNA 0 new 1 CCC r A AA A C 3 5 wysiwygFhELQMEM I mBme7htm i r 395 011431 Chromosome 3 end DNA The end of the telomere May 1999 The new view Telomere D loop 46 r Du 1 e 1 pex DNA bmdmg proteins b39 din gt egTRF2 V39 v m gpmtems Telomere Repeated sequences form base pairs Grich strand CHAPTER 27 DNA STRUCTURE REPLICATION AND REPAIR LECTURE TOPIC 5 DNA RECOMBINATION DNA Recombination Occurs between molecules that have similar sequences 39 l 39snxecu mbinatign WI gt EklllJ Homologous Recombination results in Gene replacement Gene disruption 739 Homologous recombination Shared sequences IEI Recombined Gene with some different bases Fig631 Recombinase Cre a Type topoisomerase Balm limmatirJn CHAPTER 27 DNA STRUCTURE REPLICATION AND REPAIR LECTURE TOPIC 6 DNA MUTATIONS AND DNA REPAIR if ll exacted by solar rays The Dark Side of Worshiping the Sun 4 SIGNS of MALIGNANT MELANOMA LOOK FOR DANGER SIGNS lN PIGMENTED LESIONS ATALND THEgE ABCDfs OF THE SKIN 6y may 6 Signs 0 Consult your dermatologist immediately if any ofyour moles 7 or pigmented spots exhibit Color varied from one area to anoiher Shades of an and brown lilatk sometimes white red or blue Asymmetryione half unlike the other half Diameter larger Lhan 6mm as a rule diameter of pencil eraser B Border irregular scalloped or poorly Circumscnbed border MUTATIONS ARISE FROM MISMATCHED BASES IN DNA Persistent replication errors are actually only 10399 to 103910 DNA repair improves error from 103 Chemical mutagens 39 Ultraviolet light Sunlight DNA REPAIR Base excision uracil removal TT 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 His39 to His phenotype A replication error NH H H N C N W CA mismatch mutation N N NTlt ML o Cytosine Adenine rare imino tautomer AT A T A T A T ATto GC Ef 39 39 A transition mutation purine to purine Chemical Mutagen Nitrous acid HNOZ Deamination causes AT to GC transitions Adenine Hypoxanthine CA mismatch mutation HNO2 also deaminates C to U causes GC to AT transitions I Base Analog Mismatch Thymine analog 5BU I B 5BUT mismatche A to T mutation Oral Inquot O N T N Looks 5Bromouracil Guanine like 0 am tautnmer Should be A lntercalating mutagens fit between adjacent base pairs cause base insertions leading to translational frameshifts HsRNCH N NH Mridine orange 9AmineN Zgtdimetllylamino elhylacridineAtarboxamide DNA Chem39cal Adducts OCH Allaloxin B lemchvome P450 reacts withN7 of Guanine and forms 0 covalent link OCH 3 Active DNAmodifying agent DNA Repair 3 types Altered base 3CH3Adenine 1 Base excision repair EESE39EKCiSiDr I repair pyrimidine dimers Repair Of A 39 quotquotquotquot m lI39H Ei Htideextisinn TT dlmers Remove several bases repair 3 Excision repair TT dimers adjacent bases on same strand of DNA Thymine dimer Sun ght UV light causes TT dimer formation Repair of TT dimers Cut TT CLquot 539 t x 3quot 7 r 7 l I Excisinn If a 39 IE r lutlE livdE EnigmaIt eXCInUCIease by HUMEC exdnudlease qu OH P l I l l l l l I II I l I DNA gyntheaia by DNA polymerase I Pol V 5 V I r V I 3 F I I I I I II I v Pol r 717 I lcinI39IiI IT bf LIgase DNA base E 5 i 3 c4 NH2 to c4 c0 Replica on 35 Uracil DNA Jgh osidase Remove uraCIl gt 0 gt 9 T 4 a a n AP en dun uclease P hosphod iester 1 Cut 3 5 bond pi Repair of uracil in DNA gt Iquot i uracil would lead to C to T transition JDNA polymerase POI I Ligase DNA ligase 1 r9 I Mismatch Repair Occurs soon after a DNA replication error I 3 Mismatch gt 5 Temp39ate quot39 Wm NoCH3Aon IIHIHHHITHHI newDNA Exonuclease lExonud ase 1 Endonuclease Upt02000 quot bases removed W ULL Synthesize again Triplet repeat expansions in eukaryotic DNA Associated with neurological diseases Loop lets red strand get longer with 3 more repeats added CA 0 G Cc PACKth A A 0C I l I CC CAQCAQCAQCAGCAGCAGCA CAG GTCIGTCIGTCIGTCGTCGTCGT 4 4 4 l l I 0 r00 7 A B Hismutants gt His revertants RIM 11 7 F CHAPTER 27 DNA STRUCTURE REPLICATION AND REPAIR SEE KEY CONCEPTS P1 ONLINE LECTURE NOTES
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