Class Note for BIOC 461 with Professor Bourque at UA
Class Note for BIOC 461 with Professor Bourque at UA
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Date Created: 02/06/15
Page 1 of 30 Chapter 27 Notes Biochemistry 461 Fall 2005 CHAPTER 27 DNA Structure Replication Repair and Recombination LECTURE TOPICS 1 DNA STRUCTURE 2 DNAPROTEIN INTERACTIONS 3 DNA TOPOISOMERASES 4 DNA REPLICATION The process 5 DNA RECOMBINATION 6 DNA MUTATIONS 7 DNA REPAIR KEY CONCEPTS 0 DNA structure is dynamic It can be bent kinked unwound and have different helical structures 0 Proteins interact with DNA in all biological activities involving DNA 0 DNA must be unwound to replicate 0 Topoisomerases catalyze changes in supercoiled state of DNA 0 DNA replication has three distinct phases initiation elongation and termination Termination is different at telomeres of eucaryotic chromosomes 0 DNA replication is very accurate 1X10398 mistakesbase 0 DNA molecules can recombine if they have similar sequences 0 Mutations have several causes and involve base sequence changes 0 DNA repair corrects errors using highly evolved correction systems Page 2 of 30 DNA STRUCTURE Models vs Xray Structures Static vs Dynamic Structures BDNA MODEL vs XRAY STRUCTURE WatsonCrick orhelix BDNA structure very regular came from model building based on xray diffraction data from DNA fibers consisting of parallel oriented DNA molecules REAL BDNA STRUCTURE Xray structure of crystals of 12 bp DNA dodecamer looks mostly like WatsonCrick Bform helix but there are some irregularities compared to WC model 1 L l quotAM WatsonCrick Model Real Xray structure of BDNA 36 turn per base pair 2842 turn per base pair paired bases in same plane propeller twisting bases not parallel adjacent base pairs parallel base roll structural regularities not dependent on structure details sequence specific base sequence Provides 3D uniqueness for protein DNA interactions 0 BDNA structure is dynamic The helix can be bent into an arc or supercoiled with little effect on structure BIG bend figure This permits circle formation wrapping ofDNA around proteins and packing in a cell DNA can be kinked exs AAAA39s in a row or protein binding 0 Maior and minor grooves occur in BDNA because glycosidic bonds of a base pair are asymmetrically oriented with respect to the helix axis Fig277 0 Different functional groups are located in these grooves These groups are either hydrogen bond donors d or hydrogen bond acceptors a 0 The major groove is more accessible for high specificity interactions with proteins and chemical reagents being physically much larger and having more functional groups than the minor groove Page 3 of 30 DNAB Structure Mam groove Major groove gmuv Minor groove d 39 DNAB BIG BEND Page 4 of 30 DNA bases not in same Elane Progeller twist Fig279 Propeller twist Ma39or and minor grooves Fig277 Hdonors in blue Hacceptors in red Major groove side Major groove side Minor groove side Minor groove side AdenineThymine GuanineCytosine Page 5 of DNA can exist in several structural forms Three A a and Z are discussed 3E TABLE 17 comparison n A 3 and ZDNA Ba pain poi nnn ufhclix Pitch ptr turn them W m of hm Wire innn nannai in helix axis Major groove Narrow and vcry deep Very hi mad ind nhallow Mnnn gnmvc 1w 54A 1quot Vide and quite deep Nnirnn and quite deep Helix type A B 2 Shape Broade Intermediate Nennneet R152 per base pan 2 a A 3 4 A 3 R A Helix diameter u A 23 7 A is 4 A Screw sense Righthde Rightrhanded I c rhanded Glycosidic bond mm mm aim hating aim and syn 12 i s r ix 9 n Flax ry narmw and deep Page 6 of 30 MAJOR FEATURES OF A and ZDNA STRUCTURES See Figs and Table 271 ADNA STRUCTURE A DNA is orhelical double helix righthanded Wider and shorter per turn ofhelix than BDNA Base pairs tilted 19 not perpendicular 39om axis ofhelix Most differences between A and B helix are due to different pucken39ng of sugar C39 3 endo in A DNA C392endo in BDNA Figs 317 319 The 239 OH of ribose in RNA won39t t in a quotBDNAquot type structure steric hindranceFig 276 Page 7 of 30 0 RNARNA double strands like in RNA hairpin loops and RNADNA hybrids are Aform A and Z DNA s compared to BDNA Figs274 and 2710 Top view Side View ZDNA STRUCTURE Anotherform of DNA found in DNA with alternating GC base pairs GCGCGC Fig 319 It is a lefthanded helix with a zigzag phosphate backbone and only one deep groove ZDNA is favored also by CSmethylation of cytosine which occurs in eucaryotes where evidence of ZDNA comes from binding of antibodies to ZDNA The actual biological functions of ZDNA are not known FM 14 17 253715 77 pm IWA AmtRNA Page 8 of 30 DNAPROTEIN INTERACTIONS NONSPECIFIC INTERACTIONS Deoxyribonuclease I binds electrostatically to DNA at minor groove Bonds over a whole turn ofthe helix are salt bridges between PO4 backbone and arginine and lysine NH2 groups in DNase I Since DNase I interacts with DNA PO4 groups little or no discrimination between cut sites is possible ie cuts are nonspeci c with regard to sequence See BIG BEND DNAB on p31 SEQUENCESPECIFIC INTERACTIONS In general the major groove is more accessible for high speci city interactions with proteins and chemical reagents being physically much larger and having more functional groups than the minor groove For example 0 Type II restriction endonucleases Examples EcoRV and EcoRI cut doublestranded DNA with high specificity cleaving recognition sites with twofold symmetry at identical sites on each strand These restriction enzymes are dimers of identical subunits which bind DNA with coincident twofold symmetry axes ofthe protein and the DNA recognition site NOTE See Text Chapter 9 pp 245252 for EcoRVprotein interactions Features of sequence speci c DNAprotein interactions 0 Speci c interaction is with functional groups on bases of DNA recognition sites 0 DNA is usually kinked when proteins are bound and helix is unwound allowing protein orhelices into major groove for speci c interactions 0 Speci c Hbonds between functional groups on bases on each DNA strand and amino acid side chains of enzyme orhelices or loops from Bturns are formed quotDipolarquot helices of EcoRl also interact electrostatically with the P04 backbone of DNA Page 9 of 30 Eco RV restriction enzvme interactions with its DNA recoqnition site 0 Eco RV recoqnition site has twofold svmmetrv Fig937 T A B I S39W GATATC IvMT 2539va CTATAG WS 3 Symmetry axis INT AACTQNS M9 MH SOR WW5 Flu uni inanaamnnao7wz Siryui Biuzhammlm mm mm a 1995 My w H mm inn minyiy Cm 52 0 Eco RV as mmetric dimer binds on one side of the DNA helix Fig 3110 4th Ed Page 10 of 30 Eco RV opens the DNA helix Fig 3110 4 h Edand kinks the DNA about 50 Fig940 Specific Hbonds form between functional groups on bases on each DNA strand and amino acid side chains of Eco RV Bturns Fig939 A 75 3239 y Gly184 y 182 1 6 L r Thymine Adenine Eco RV has four structural elements that are evolutionarilyconserved being also found in other Type II restriction enzymes Fig944 Page 11 of 30 Eco RI restriction enzvme interactions with its DNA recoqnition site Eco 2 flung m39im ML 0 6 Hbonds with amino acids 5 pbJ Mk5 4d ETT C Q iU n3 gnu W DNA backbone P E K 3 g k w 0 Dipole charge nrtgrgitions 8 I a a Helix P70 n 39 CH 0 Hbonds with H 3 amino acids Page 12 of 30 DNA TOPOLOGY DNABINDING PROTEINS ALTER THE TOPOLOGY 0F DNA UHWOUnd Ovenwound O Negative supercoiled circular DNA is compact and is energetically favored Most DNA in cells has negative supercoiled righthanded superhelices Superhelices are underwound This facilitated DNA helix unwinding for replication recombination transcription etc 0 Positive supercoils lefthanded make opening the helix more difficult O The topology of DNA state of supercoiling can be changed by unwinding or winding supercoils Changes in linking number result in different DNA topoisomers Changes require cutting one or both DNA strands Figs272 3116 3117 3118 Different states of DNA su ercoilin negative and positive Topo isomOMSes Change 95 0 full ag39epp 9 A misommsa Relaxed DNA Highly supercorledr DNA 5quotquot 35min Page 13 of 30 Topisomerase enzymes can DNA convert to supercoils 31 Relaxed DNA Lk 20 Positive supercoi 1 Li 18 supercoil Lk 22 Topoisomerase 2 strands cut righthanded supercoils DNA Gyrase uses ATP Topoisomerase 1 strand cut lefthanded supercoils NOTE Helicase in DNA replication adds positive supercoils makes NO cuts and uses ATP DNA TOPOISOMERASES Topoisomerase catalyzes relaxation of negative supercoils by 1 cleavage of one DNA strand 2 passage of a segment of DNA through the break and 3 resealihg the break No ATP energy required for this reaction A 539P at cleavage site is activated by covalent linking to a tyrosine on the enzyme Then a 339OH hucleophilic attack ligates the cut strand of DNA after removing one or more supercoils Fig 2722 formay this is adding positive supercoils Page 14 of 30 Topoisomerasel cuts one strand cuts rotates ligates Fig2722 O Topoisomerase ll DNA Gyrase in DNA replication A class of proteins that add negative supercoils to DNA using ATP hydrolysis for energy 9 kcalmole Fig2724 In replication 200bp of DNA wrap around the gyrase holoenzyme molecule ATP is bound and each strand is cut staggered cuts and covalently linked to different tyrosines to quotanchorquot the DNA Then a segment of DSDNA passes through the cut the cut ends are religated and ATP hydrolysis releases the DNA from the gyrase Two negative supercoils are added with each catalytic step as a result of DSDNA passage through a break in both strands Two DNA gyrase inhibitors are nalidixic acid prevents strand cutting and rejoining and novobiocin blocks ATP binding are a segment r segment Page 15 of 30 DNA ligase joins free 339OH ends with a 539P group of adjacent bases can think of as a quotnickedquot DNA strand forming a new phosphodiester bond J P i H r 10f mck39 W DNA 51 757 EVA wand i imam 7 AMP DNA smr 3 u i i 5397 DNA 5 New 339539 phospodiester bond NAD or ATP react with DNA igase to give an enzymelinked AMP that is then transferred to the 539P end of the DNA This AMPactivated 539P is subjected to a nucleophilic attack by the free 339OH end AMP is the leaving group and a new 339539 phosphodiester bond results Two high energy Pbonds are used in the complete reaction with either NAD or ATP Fig 3112 and p7612 of 5 h Ed Page 16 of 30 DNA REPLICATION The polymerases Iquot Review Ch5 Figures pp 1315 DNA POLYMERASES IEcoli examples Pol land Pol Ill 0 DNA POLYMERASE IPol I HAS THREE DIFFERENT ACTIVITIES 1 Tem lateDirected Pol merase activi 539 to 339 Fig522 Synthesizes DNA from 539 to 339 E coli Pol enzyme is simplest and best understood DNA polymerase Though not responsible for most chromosomal DNA replication it has functions in DNA replication and DNA repair A 103kd monomeric a ZN enzyme A Processive enzyme ie don39t need associationdissociation step for each nucleotide added About 20 bases added before enzyme falls off DNA and another one takes its place Error rate 1 x 10 Primer swam Primer sum puele amdwal vNa pug aman mm 5 2 Proofreadin 339t0539ExonucleaseActivi 1 A t T 0 Hydrolyses nucleotides in 339 to 539 direction at quotquot4quot quot399 WWW 339OH end of DNA primer if wrong nucleotide I quot39A Hymn sh Lanna 0 Editing proofreading activity helps prevent 95 TA errors during DNA replication 0 If a mutation causes reduced proofreading get higher mutation rates if increased can reduce mutation rates Misses wrong base D at rate 1 x 10 39 glue buts Page 17 of 30 3 ErrorCorrecting 539 to 339 Exonuclease Activity 939 3 Fig 31 25 I quotquotd l O Hydrolyses DNA from 539 to 339 direction if a 39 free 539P end of a doublestranded DNA is 7 a encountered O Cuts are at 539terminal nucleotide or several nucleotides from 539end Corrects errors in preexisting DNA Hydro y site 539 339 nuclease octivilv O Involved in repair of DNA and in removal of RNA primer used for DNA replication Place conce t of DNA ol merase accurac error rates in context of Flow of genetic information Review Ch5 Part2 Figures p21 Central Dogma Pol Active Sites and Structure DNA polymerase I has a different active sites for each activity 4 Ed Figs 3126 3127 and 5quot Ed Figs271115 539 to 339 Polymerase domain 543539 ralvnerau sibe Page 18 of 30 Role of two Mg in Pol 539 to 339 polymerase is to coordinate 339 OH with dNTP and with 2Asp of Poll Fig2712 Correct basepairing of incoming dNTP is anchored by Hbonds between minor groove Hacceptors same place for AT and GT and Arg and Gln of Pol Fig2713 Correct dNTP binding induces conformational shift that gives tight binding pocket for dNTP and template DNA Fig2714 Page 19 of 30 0 339 to 539 Exonuclease activity of Pol I when added base is released from polymerase site and edited at distant site Fig2715 gt Migraliou m Template exunudease 5le Strand Exonuclease active site 0 DNA POLYMERASES H AND quotI E coli Mutants lacking Poll grow and replicate DNA normally DNA polymerase II function unknown and I found 15 years after Pol Both have per cell lower number of molecules and activity than Pol Both have 539 to 339 DNA polymerase and proofreading 339 to 539 exonuclease activity but no 539 339 exonuclease function 0 DNA POLYMERASE lll catalyzes most of E colichromosomal DNA replication It is a multisubunit asymmetric dimer enzyme It adds deoxyribonucleotides at a rate of 1000 basessec to the primer This is about 100 times faster is more processive than Pol thousands of bases compared to about 20 The Oi subunits are catalytic while a B2 dimer forms a sliding clamp with a hole in the middle which can accomodate the DNA templateprimer complex Figs2730 31 DNA binding site Page 20 of 30 DNA Polymerase lll e dimer DNA Polymerase lll DNA Complex STUDY HINT COMPARE PROPERITES OF DNA POLYMERASES I II AND III DNA REPLICATION The process INITIATION OF DNA REPLICATION 0 Replicating DNA molecules appear to be in 6 theta structures showing E coli DNA as circular during replication DNA unwinding and replication takes place at 2 replication forks and proceeds in both direction simultaneously 0 Replication starts at a unique site OriC 245 bp from two replication forks Fig2725 Binding of dnaA protein and othersinitiates replication Tandem array ol isimer sequences AT rich Consensus sequence Page 21 of 30 THE DNA REPLICATION CYCLE Replication is complex Many proteins in addition to polymerases are involved One replicating DNA strand leading strand is synthesized continuously and the other lagging strand is synthesized discontinuously The processive Pol III dimer connects about 1000 basesFigs273 and 27 Apparent directions of Actual 539 to 339 direction of DNA synthesis DNA synthesis Replication 5 Leading strand Parental fork 539 Parental DNA DNA 5 W 1 I 5 Okazaki fragments 3 3 2 3 3 Lagging strand LEADING STRAND SYNTHESIS elongation o Helicase unwinds DNA ATP hydrolysis required introduces positive supercoils O SSB protein binds to the parental single strands as they are unwound 0 DNA gyrase introduces negative supercoils to relieve torsional strain ATP hydrolysis required Page 22 of 30 0 RNA primase a specific RNA polymerase synthesizes a primer of about 5 bases long The RNA primer is later removed and the gap filled in by Poll Fig2726 DNA template 3 5 iPrimase RNA primer iDNA polymerase III New DNA 0 Pol III dimer adds deoxyribonucleotides to the RNA primer TERMINATION OF DNA REPLICATION WM Pol l cleaves off RNA primers and fills in 1 3 gaps both leading and lagging strands L DNA ligase seals gaps Fig 3138 Cwnlsiw durum arm ohm k Ft MM F 39YG MEA Page 23 of 30 LEADING AND LAGGING STRAND SYNCHRONOUS SYNTHESIS Figs 2732 33 Helicase Singlestranded binding protein 539 Leading strand LAGGING STRAND SYNTHESIS elongation The lagging strand appears to form a loop to reverse polarity presenting itself to one subunit of the Pol III dimer in the correct polarity for DNA synthesis A er connecting about 1000 bases a new primer is needed for another 1000 base fragment etc N 51 3 Lagging gt strand Page 24 of 30 Termination of Eucaryotic DNA replication The Problem it s a linear chromosome so how to complete the ends Can t just ligate ends and get a circle as with E coli chromosome see Fig 3715 4th Ed Eucaryotic Telomere structure Fig2735 Grich strand DNA replication leaves one incom Parental DNA incomplete daugmer Complelc daughter plete end Fig 3715 5 5 350 5 Ramanun gnu 3 339u s lagging strand mm mm Lemma strand 5 3 arm 539 Erasure am 5 2v 5 a vu z 3 uv s39 4 elomere s nthesis b telomerase Fig2736 Telomzll ff In 70 339 my A L t L AAC Telumerase RNA x r Elongnnu n 1 5095 I GEGT I l 1 I Q m Aucml 0H3 iramlocaxzun VUH Immmccrmccol 539 ALUCAAC L a DNA RECOMBINATION Recombination mechanism Cre recombinase and Hollidav Junctions Fig631 and Figs 2737 39 Band formaiion Recombination gt Page 25 of 30 A Targeted gene Mutated gene B 39 A A g g Mutation in the targeted gene Holliday junction Isomerizalion Page 26 of 30 MUTATIONS are DNA base changes that arise from mismatched bases in DNA caused by 0 replication errors chemical mutagens ultraviolet light sunlight SPECIFIC MUTAG ENS Base analogs cause mispairing and transitions chemicals that modify bases cause transitions intercalating agents cause insertions or deletions errors ARE MUTAGENIC AGENTS also CARCINOGENS o Ames test Salmonella His39 to His Rare CA base pair N Hgt H N N N w Nlt m39H N iN O N Cytosine Adenine rare imlno tautomer Chemical muta ens Nitrous acid deaminates adenine get AT to 60 transitions Nitrous O add 139 Cytosme r g H Adenine Hypoxanthine N f f K WinsN N ultraviolet light causes pyrimidine dimers then get replication H thl H O Base analog 5BU Br OH NH gt w NWlt NNampN O H w H 5Bromouracil Guanine enol tautomer Aflatoxin activated bv cvtochrome P450 and adds to N7 of guanine Causes GC to TA transversions Thmine dimers adjacent bases in same DNA Page 27 of 30 Intercalatin a ent causes insertions frameshifts CH3 I w CH3 N CH3 Acridine orange OCH3 Aflatoxin B1 letochrome P450 Active DNAmodifying agent strand Thymine dimer Page 28 of 30 DNA REPAIR replication errors are 1 in10E but fixed by repair to 1 in 101 bases Repair pathways Fig 27 47 aaseexcision iepair jW4 wwvm mow NW epail TT dimerrepairExciSion of iesion U dimers new DNA synthesis by Poi i and iigase to seai nick Xeroderma pgmemosum is caused J Excision oi a I2riiudenlid2 lrdgmem by quotwas Exdnudease by a defect in me exonuciease or any of 8 oiner genes Wmcn removes pynmidi e dimers Fig 27 49 m mm mm symhcsis by DNA DonLrase I iniiis oi om Kigase W O uracil removal uracil comes from deamination of cytosine in DNA is by speci c uracil Nglycosidase which leaves thymine alone no cleavage Thus the CSCH3 group of thymine may allow discrimination of it from the deamination product Uracil of cytosine which must be removed This is another DNA delity enhancer Fig 3144 Mismatch l Page 29 of 30 lll AQA TUT lll Umdl DNA glycosaas lll lll 5 i Jl Jnmpoymm 4 DNAligaw M c A c r L 4gt o mismatch repair Mismatch repair in Ecoli is carried out by several proteins which are C l related to human proteins in which mutations it lT lExonuclease l G l DNA polymerase in c l l I l l C l l l l are related to incidence of colorectal stomach and uterine cancers Fig 3145 Page 30 of 30 Trigletrepeats occur m eucaryouc chromosomes m tandem arrays Addmon of more than norma number ofmem cause geneuc dwseases Exs Hurmnglon dwsease other neuro ogwca dwseases known 00 ng 27 52 CA 0 0 CC FQCquot A A 09 cc CAGCAGCAGCAGCAGCAGCA mcmcmcmcmcmcm t 06 or
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