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Discwk2 - Notes for lecture 3 ­6 GSI: Nikki...

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Unformatted text preview: Notes for lecture 3 ­6 GSI: Nikki Definitions: Processivity—how far (e.g. how many nucleotides) a protein (e.g. polymerase) can go (add) before dissociating from the substrate.  ­Tight interaction with the DNA at the palm (active site) can increase processivity  ­The sliding clamp is a processivity factor Fidelity—accuracy of DNA replication  ­Tightness of fit in the active site of polymerase can increase fidelity  ­Amino acids at the active site can discriminate mismatches and RNA (2’OH)  ­Exonuclease activity of replicating polymerases also ensures accuracy Semi ­conservative—the product of DNA replication retains one parental and one daughter (new) strand. Polymerases: 4 required components for function: template (DNA to be copied), dNTP (ACTG), Mg2+ (for stabilizing intermediates in the Pol active site), and primers  ­ ­> all DNA polymerases need these, especially primers  ­ ­> primers have 3’OH for Pol to attach new nts and also ensure accuracy by base stacking with the template PolI 3’ ­>5’ exo, 5’>3’ exo, pol activities are all in one peptide Slow Low processivity Mainly proofreading PolIII 3’ ­>5’ exo; 3 peptides; dimerize Fast replication Highly processive RNA primer Euk Pol δ and ε cooperate in replication, both have 3’ ­ >5’ exo activities Fast replication Highly processive RNA/DNA primer PolI  ­Mutants at the active site are viable  ­5’ ­>3’ exonuclease: cuts the backbone of a duplex to make ssDNA, perfect for primer removal (nick translation)  ­3’ ­>5’ exonuclease: proofreading, takes turns with Pol when mismatches are detected at the Pol active site; the 3’OH of the growing chain flips to the exo active site (faster kinetics) because 3’ ­>5’ exonuclease likes ssDNA, and the polymerase doesn’t. Replication steps and the proteins involved: 1) Helicase: loads first by DnaA and DnaC onto ssDNA  ­ polarity can be 3’ ­>5’ or 5’ ­>3’ (DnaB in E. coli; bound around lagging strand at the replication fork). e.g. MCM, the helicase in eukaryotes is 3’ ­>5’, so it’s on the leading strand at the fork.  ­ ATP ­dependent  ­ hexameric—this creates movement: each subunit takes turns to contact DNA.  ­ Helicase interacts with and stimulates primase (DnaG in E. coli) 2) SSB proteins: required to keep unzipped DNA duplex separated  ­ binds each other and DNA; the latter is stretched like a coil of wire from 3.6 angstrom per turn to 5  ­ cooperative binding, fits each other so no nucleotide is free.  ­ mainly important for the lagging strand, there should be some on the leading strand, too, because replication of both strands are coupled. 3) Primase: recruited by helicase  ­ can start de novo synthesis, no need for 3’OH and not sequence specific  ­ not accurate nor processive, which is good, so there’s not too much clean up to do later to get rid of the primer that might contain mismatches  ­ NTPs instead of dNTPs Prokaryotes Eukaryotes Primase + Pol α DnaG Short RNA primer Primase lays down RNA primer, then Pol α lays down a DNA primer using the 3’OH on the RNA primer Because PolIII can tolerate both A and B Pol δ and ε can only tolerate B form of forms of DNA duplex; A form is wider, DNA; tighter grip for more accurate thus PolIII has slightly loosened grip, not replication since euk genome is much as accurate as Euk Pol bigger 4) Clamp/loader complex  ­ Clamps form a ring shape; bacteria’ β clamp is a dimer and eukaryotic PCNA is a trimer  ­ Clamp can’t just open and load onto DNA on its own because then it could open while on the DNA and fall off, defeating the purpose of being a processivity factor: requires a loader complex (γ complex in bacteria)  ­ β clamp has the same binding site for PolIII and loader complex; this competition between the two binding leads to equilibrium (it actually favors binding to PolIII) Closed clamp around primer/DNA duplex also drives hydrolysis of ATP and thus dissociation of the clamp loader, further favoring binding of PolIII  ­ The loader complex recognizes the 3’OH of RNA primer Loader complex actually binds 1 monomer of the beta clamp dimer better (this was measured from mutagenesis studies; i.e. when 2 residues on the monomer interface are mutated so clamp cannot form a ring, loader binds to monomer with 50x higher affinity). So loader can force open the ring once bound to a monomer in the favorable ring conformation After replication 1) DNA ligase: eukaryotes and viral ligases require ATP; E. coli DNA ligase uses AMP derived from NAD+.  ­ ­> Seals nicks in the backbone of one strand in a dsDNA, they don’t anneal two separate strands into a duplex.  ­ ­> T4 ligase can seal nicks in the backbone of both strands in a dsDNA 2) Primer removal Prokaryotes PolI’s 5’ ­>3’ exo chews up primer as the polymerase domain lays down new DNA in the 5’ ­>3’ direction RNaseH is used as a back up, it chews up specifically RNA in an RNA/DNA hybrid (i.e. primer/template hybrid) Eukaryotes Pol δ and ε displace primer as they continue to polymerize, thus leaving a 5’ flap FEN I recognizes the 5’ flap; it’s an endonuclease RNase HI can also chew up the primer as a back up. Experimental application: You want to use a fully purified and reconstituted DNA replication system (in vitro) such as below to test some conditions: 5ʼ 3ʼ 5ʼ 3ʼ You can only detect replication products in a phosphoimager because dNTPs are 32P labeled. For each experimental condition, draw out what the products would look like. (Answer on the next page) Lane 1: Include all components in the replication reaction Lane 2: Leave out ligase Lane 3: Leave out helicase Lane 4: Leave out primase Lane 5: Lave out the clamp loader Lane 6: Leave out ATP 1 2 Wells to load the sample 3 4 5 6 5ʼ 3ʼ 55ʼ ʼ 5ʼ 5ʼ 3ʼ ʼ 3 $8(+$9:;$2+1/36)83&5$12&-'[email protected]$/)D+/+-#$.&2$+)6($+<1+23,+58)/$ 3ʼ +/$&.$8(+$9:;$2+1/36)83&5$12&-'[email protected]$/)D+/+-#$.&2$+)6($+<1+23,+58)/$ +/$&.$8(+$9:;$2+1/36)83&5$12&-'[email protected] ABC$/)D+/+-#$.&2$+)6($+<1+23,+58)/$ +58$/)5+7$35$8(+$?+/= AB 3ʼ +/$&.$8(+$9:;$2+1/36)83&5$12&-'[email protected] C$/)D+/+-#$.&2$+)6($+<1+23,+58)/$ 3..+2+58$/)5+7$35$8(+$?+/= -3..+2+58$/)5+7$35$8(+$?+/= -3..+2+58$/)5+7$35$8(+$?+/= +-$?+/$&.$8(+$9:;$2+1/36)83&5$12&-'[email protected]$/)D+/+-#$.&2$+)6($+<1+23,+58)/$ +$6&,1&5+587$35$8(+$2+1/36)83&5$2+)683&5 3ʼ //$8(+$6&,1&5+587$35$8(+$2+1/36)83&5$2+)683&5 )//$8(+$6&,1&5+587$35$8(+$2+1/36)83&5$2+)683&5 $35$-3..+2+58$/)5+7$35$8(+$?+/= <1+68+-$?+/$&.$8(+$9:;$2+1/36)83&5$12&-'[email protected]$/)D+/+-#$.&2$+)6($+<1+23,+58)/$ )//$8(+$6&,1&5+587$35$8(+$2+1/36)83&5$2+)683&5 7+ $/3?)7+ 8$/3?)7+ &)-+-$35$-3..+2+58$/)5+7$35$8(+$?+/= Wells to load the sample -+$)//$8(+$6&,1&5+587$35$8(+$2+1/36)83&5$2+)683&5 8$/3?)7+ 1 2 Wells 3toloadthe sample 5 Wellsto load the sample the sample Wells to load 4 $(+/36)7+ 36)7+ 8$(+/36)7+ $356/'-+$)//$8(+$6&,1&5+587$35$8(+$2+1/36)83&5$2+)683&5 $&'8$/3?)7+ Wells to load the sample 8$(+/36)7+ 2 3 4 1 2 33 44 55 5 1 2 $/+)*+$&'8$/3?)7+ $123,)7+ 8$123,)7+ ,)7+ Wells 3 load the sample 5 to 2 4 $&'8$(+/36)7+ 1 8$123,)7+ Wells to load the sample $/+)*+$&'8$(+/36)7+ 1 2 3 4 5 $8(+$6/),1$/&)-+2 8$8(+$6/),1$/&)-+2 $6/),1$/&)-+2 $&'8$123,)7+ 1 2 3 4 5 8$8(+$6/),1$/&)-+2 $/+)*+$&'8$123,)7+ epolymerase will be polymerase will be $&'8$8(+$6/),1$/&)-+2 merase will be d o you will get bands ndssoyou will get bands e ou will get bands $/+)*+$&'8$8(+$6/),1$/&)-+2 ly polymerase will be y a smear) indicating blya smear) indicating ndhat polymerase bands so you will went erthat the pol IIIgetwill be s tthe the pol III went smear) indicating bly a smear) will get bands p and so you ve.loader the polymerase will be Ahe pol III indicating to.cessive andwentwent get bands t that the pol III will so indicating s robably a smear)you izes (probably a smear) indicating Aces n. III $;IC 8$;ICthat the polthe went went nt NA. pol III D distances that off the 8$;IC DNA.you onʼtʼtwork, so you on work, so $&'8$;IC up to an replication hanreplication up to $/+)*+$&'8$;IC 5ʼ 6 66 6 6 6 6 Full length DNA pieces, Full length DNA pieces, ully replicated fully replicated Full length DNA pieces, Full length DNA pieces, fully replicated DNA pieces, fFull Full length pieces, ully length DNA replicated TP, the clamp oArk, so you you onʼt the clamp also oATP,work, so also merase less less ase wonʼtisup to up to tymerase iswonʼso you so you han replication plicationwork, t work, e helicase aasewill replication up to mATP, the than1replication up to ger clamp alsoless ohe longer clamp also t se willhave hingthan have 1 less er noWith is less the also also erwith (since clampisclamp with (sinceATP is RNA). ith lymerase no ATP, fork. ATP, the ATP RNA). se is less b so the up is made have ieepolymeraseto less is less mase willupto 1 less d,emade polymerase willrimase 1Ainto the less have less rporate anwill have 1 is RNA). rporate (since ATP new ,p addition, primase the new mer withanA intowill have 1 less thwillbe with (since (since RNA). (since ywillmade ATP is RNA). ebprimer shorter withATP is ATP is RNA). ake the shorter than e be primer than up to de be rimers made made primerswith A into up to onlyup toan less ers can only beup to the new orporatewithless t tan to incorporate of the daughter eding A into the into A daughter ni oothem,shorter new thenew them,so some an tencorporate an A thantheinto the new y will be so some of average, they shorter shorter than than the length ,rtthey the be will be to the ehan will length up to the shorter than primers with lessup than leeIʼIm assuming that you get shorter with less rrterʼm lessprimers with less s withassuming that you daughter nII oprimersso some of the t adding on to them, so pIike in problem 9some some of the daughter like to them, ilelm, soproblemthem, soof the daughter ng on in some so9the daughter of so r than the length the to the than up ssociates, itprobably ociates, sorter thanitthe length length up heven shorterprobably up to the to the re IOTE: Here Iʼm assuming that you tmem assuming same . hestrand at the that you N ʼ length up to the me strand at the same a Here Iʼm assuming that you : p- like in problem 9 in so imental setup like in problem assuming that you 9being9 so g -this is important this is in problem so setup likeimportant in being lymerase dissociates, it issociates, it9 so in problem probably probably sive replication. sive replication. probably e dissociates, it iate to the same strand at the ae same strandthethe same same me strand at at tue replicating - this is important in being es, it probably same h g - this icating atthis issame in being trand - is important in being the important non-processive replication. Oakazaki sive replication. ocessive replication. s is important in being plication. 3ʼ fully freplicated ully replicated Replication up to Replication up to tthe fork he fork Replication to Replication upup Replication up to to Replication up to he the fork fork he tfork t the fork Fragments ragments Oakazaki Oakazaki Oakazaki Fragments FFragments ragments Oakazaki Fragments Lane 1: Fully replicated full length DNA Lane 2: Okazaki fragments that are not linked together; some full length from the leading strand Lane 3: Replication can only go up to the fork without helicase to further unzip DNA Lane 4: No replication occurs because DNA polymerases cannot start de novo synthesis without a 3’OH Lane 5: With no clamp loader, the polymerase will be much less processive so you will get bands of different sizes indicating the different distances that PolIII can go before falling off (a smear) Lane 6: No ATP, thus no helicase, no clamp loader, and no ATPs for primase to incorporate to incorporate into a primer. Therefore you can only get replication up to the fork but with shorter primers and a unprocessive PolIII smaller bands ...
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