Exam 3 Notes
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Date Created: 10/07/15
DNA Replication DNA Replication is semiconservative 3 proposed models of DNA replication a Semiconservative model b Conservative model DNA stays intact and acts as a master copy AKA Xerox model Master slave model A lot of proponents and biological examples WatsonCrick Base pairing MeselsonStahl experiment The most beautiful experiment in biology c Dispersive model Fragmented Copiesparentals are dispersed No real mechanism proposed as to how this might occur 0 Experiment to determine which of the models of DNA replication was correct 0 Experiment 0 Grow E Coli in medium with 15N nutrients so each atom has a higher density so that the products of these cells will have a higher density 0 Nutrients replaced with 14N Every 30 minutes the doubling time of bacteria the cell was removed E Coli taken out of media broken up DNA extracted and isolated Chromosome being broken up DNA mixed in 6M cesium chloride CsCl salt higher solubility than NaCl This mixture was then placed in an ultra centrifuge which develops a density gradient by centrifugal force This separated the different isotopes into bands and then you could infer from the isotope ratio how much of your DNA was parental and how much was newly synthesized I This was essentially determining after each round of replication how much of the DNA was parental 15N and how much was newly synthesized DNA MN I Each double stranded DNA will show a single band So if it s double stranded DNA with one strand parental and one strand newly synthesized such as in semiconservative model you would see one band at 145 Actual results seen semiconservative model Cycle 1 Time 0 minutes DNA all 15N all One band at 15 parental Cycle 2 Time 30 Heavylight hybrid One band at 145 minutes DNA Cycle 3 Time 60 One heavylight 2 bands One at minutes hybrid and one 145 and one at lightlight 14 Cycle 4 Time 90 3 lightlight to one 2 bands One at minutes heavy light hybrid 14 one at 145 31 lighthybrid ratio Difficult to do more than 4 rounds because replication becomes unsynchronized Theoretically round 5 would show 1 intermediate to 7 light Round 6 would show 1 intermediate to 15 light Conservative model ruled out after cycle 2 because there is 1 intermediate band Conservative model would show 2 bands one at 14 and one at 15 because one would be entirely parental and one completely new Dispersive model ruled out after cycle 3 because dispersive would show an intermediate at about 143 If DNA was resuspended in urea a denaturantcauses the strands to separate you could check between dispersive and other models DNA Polymerases 1955 Arthur Kornberg identified enzymes necessary for DNA replication in E Coli 0 Fractioned E Coli and supplied each part with all things necessary for replication except polymerase O Whichever fraction could successfully replicate contained the polymerase O Kept fractioning until narrowed it down to a single polypeptide 0 What he identified was Pol I from gene polA Thought it was the only polymerase 0 Not the polymerase he was looking for could knock out the polymerase and still replicate 0 Led to the discovery of Pol 11 After isolation of Pol I discovered that there are 5 components necessary for DNA synthesis 0 All four dNTPs precursors for the nucleotide phosphatepentose sugarbase DNA P01 1 template DNA Primer a short DNA chain need to startprime the synthesis reaction Magnesium ions Mg2 which is needed for optimal polymerase activity All DNA polymerases catalyze the polymerization of dNTPs into a DNA chain 0 At the 3 end of the chain DNA polymerase catalyzes the formation of a phosphodiester bond between the 3 OH group of the deoxyribose of the last nucleotide and the 5 phosphate of the incoming dNTP I The energy for the formation to occur comes from the release of two of the three phosphates on the dNTP I Important concept the lengthening DNA chain acts as a primer in the reaction 0 At each step in lengthening the chain DNA polymerase finds the correct dNTP that can form a complementary base pair with the nucleotide on the template strand I Nucleotides are added at about 850second in E Coli and 6090second in human tissue culture cells 0 DNA polymerase is processive meaning that it moves along the template continuously doesn t dissociate and reconnect 0 5 DNA polymerases have since been identified in E Coli O I and III are necessary for replication I II IV and V are involved in DNA repair Pol Polymerizatio Exonucleas Exonucleas Molcell Encoded n e e by gene 5 to 3 3 to 5 5 to 3 I single Yes Yes Yes 400 polA subunit II single Yes Yes No polB subunit III multi Yes Yes No 1020 10 genes subunit complex 0 3 5 exonuclease activity meaning that they can remove nucleotides from the 3 end of a DNA chain used in error correction in a proofreading mechanism Immediate proofreading backspace button When incorrect base is inserted reduces error frequency of about 106 to 109 0 5 I 3 exonuclease activity goes same way as polymerase Nick translation activity Chews at DNA upstream to remove it While synthesizing Used in repair of DNA and in discontinuous strand synthesis 0 Pol III harder to detect than Pol I less molecules per cell Pol III is the polymerase not a single subunit like the other 2 0 Pol III Initiation of Replication Replication begins at a single location called the origin of replication oriC in E Coli contains three 13bp repeats and four 9bp repeats 1 Initiator proteins bind to the origin of replication they are able to recognize specific sequences and bind Which bends the DNA Which facilitates opening 2 DNA helicase with DNA helicase loader binds to the initiator proteins and breaks bp interaction 0 DNA double helix denatures into single strands to form a replication bubble Each strand is now a template strand and 2 replication forks have formed 3 Helicase loads onto DNA and moves sequentially to break bp and unwinds DNA The DNA untWists from both sides of the replication bubble called bidirectional replication 4 Helicase recruits DNA primase forming a complex called the primosome Primase is important because DNA polymerase cannot initiate synthesis of a DNA strand they can only add to a preexisting strand So the primase makes a short RNA primer Which is later removed and replaced With DNA 5 The RNA primer is extended as a DNA chain by DNA polymerase Semidiscontinuous DNA Replication Ideally Continuous synthesis but cannot occur because the DNA chain cannot elongate in a 3 to 5 direction Actually occurs Semidiscontinuous synthesis SSB singlestrand DNA binding proteins bind to each singlestranded DNA after they are opened by the helicase stabilizing them and preventing them from reforming double stranded DNA a process called reannealing An RNA primer goes on the bottom template strand on the 5 end This will be the leading strand and will only have one primer The DNA primase then synthesizes another RNA primer on the top template strand The polymerases add nucleotides to the 3 end and displace SSB proteins as they move along the template strands Since DNA synthesis can only occur in the 5 to 3 direction the DNA synthesizes in opposite direction on the two template strands O The new strand being made in the direction opposite of the movement of the replication fork is the lagging strand Helicase untwists more DNA causing the replication fork to move along the chromosome DNA gyrase a form of topoisomerase relaxes the tension in the DNA ahead of the replication fork which would otherwise spin so fast it would burn up Replication shuts down if topoisomerase is inactive Leading strand continues synthesis Lagging strand synthesizes until it reaches the end of the segment Then a new initiation of DNA synthesis occurs an RNA primer is synthesized by DNA primase at the replication fork DNA pol III adds DNA to the RNA primer to make another DNA fragment Each fragment on the lagging strand is called an Okazaki fragment After discontinuous synthesis on the lagging strand is done Pol III leaves and Pol I binds At this point there is no gap just a nick between RNA primer and new DNA DNA Pol I simultaneously digests the RNA primer strand ahead of it 5 to 3 exonuclease activity and extends the DNA strand behind it 5 to 3 polymerase activity called nick translation DNA ligase then joins all the DNA fragments removes nicks In reality the replisome is bound to both strands and includes both of the DNA Pol IIIs the helicase and the primase Which all move together To account for the synthesis in opposite direction the lagging strand loops ips over so that all the key replication proteins can move together This is called the simultaneous synthesis model Where both strands are synthesized at the same time in different directions Rolling Circle Replication 0 Different form of replication that occurs in various bacteriophages Phage A 0 Circular DNA same problem as E Coli DNA but solves in a way other than the semiconservative method 0 1 Generation of nick at the origin of replication 0 2 5 end is displaced and covered by SSBs 0 3 Polymerization occurs at 3 end to add new deoxyribonucleotides 0 4 DNA on the other strand the displaced strand starts at the circle and goes to the end of the segment As the circle unrolls more it again starts at the circle and goes to the end of the precious strand making a 2rld Okazaki fragment 0 Rolling circle replication is thus Semidiscontinuous but is not semiconservative because it continually rolls around and eventually after one full roll it s peeling off newly synthesized and is thus following the conservative model as it synthesizes off of new DNA on the lagging strand and synthesizes off the parental in the leading strand which acts as a master copy Mitochondrial DNA Replication 0 2 origins of replication each is unidirectional initiation occurs at each at a different time 0 Opens one origin starts continuous synthesis Hits other origin starts replication in other direction Both go all the way around in different directions 0 This replication is semiconservative and continuous Eukaryotic DNA Replication 0 Replication occurs in S phase approx 9 hours 0 If eukaryotic cells synthesized like E Coli it would take about a month because the genome is so much larger 0 To compensate humans have about 30000 bidirectional origins each origin replicates about 100000 base pairs replicon or replication unit stretch of DNA from origin of rep to the termini of replication where the 2 adjacent replication forks fuse 0 Not all origins initiate at the same time if they did it would take about an hour to replicate but in reality takes about 9 hours Those that express genes synthesize first Later synthesized genes are dormant Eukaryotic polymerases 0 Not as much is known about eukaryotic polymerases as is known about E Coli Pol 0t Alpha Nuclear Repair Tetramer Initiation P01 5 Delta Nuclear Repair Tetramer Elongation Pol E Epsilon Nuclear Repair Tetramer Elongation Pol B Beta Nuclear Repair Pol I Monomer Pol v Gamma Mitochondrial Dimer Replication 0 Replicase Complexes 0 P01 0t and primase initiation 0 P01 5RFC clamp loaderclamp cDNA leading strand synthesis 0 P01 E or P01 5 lagging strand synthesis 0 RNA Primer Removal 0 RNAase HI 5 to 3 endonuclease actiVity Polymerase replaces RNA with DNA Ligase links Okazaki fragments In E Coli this actiVity is part of Pol I In Eukaryotic organisms it s separate Linear Chromosome Selective advantage of linear chromosomes crossing over recombinationl genetic diversity So advantageous that it overcomes the disadvantage of telomere erosion problem After replication there s still an RNA primer on the very end which is then removed leaving a gap at the telomere lost genetic info After multiple rounds the ends are shortened telomere erosion At ends of chromosomes repeating sequences 100s to 1000s of tandem repeat sequencesacts as a buffer against erosion Repeat sequences are noncoding regions Number of repeats limits the amount of times they can divide Somatic cells can t repair the loss of these sequences Once all the repeats have eroded the cell will sense that the DNA is damaged and shut down replication This is thought to contribute to aging Germ line cells have a way to repair this DNA so that they re not limited to how many times they can divide 0 Telomerase a reverse transcriptasemakes DNA on an RNA template binds to the overhanging 3 end of the chromosome 0 New telomerase DNA is then synthesized from the telomerase RNA template 0 This new DNA of repeat sequences compensates for the loss caused by removal of the primer
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