1Bacterial DNA replicationSummary: What problems do these proteins solve?Tyr OH attacksPO4 and forms acovalentintermediateStructuralchanges in theprotein open thegap by 20 Å!2DNA ligase IDNA ligaseLigasetopo IItopo IVDecatenationtopo I or topo IIgyraseRemove +sc at fork (swivel)RF-ASSBssDNA bindingPCNAβ Sliding clampRF-Cγ complex Clamp loaderpol δ, εpol III (α, ε , θ subunits) CorePolymerasepol α primaseFEN 1 (also RNaseH)Primase (DnaG)pol I’s 5’-3’exoPrimasePrimer removalT antigenDnaBHelicaseSV40 (simian virus 40)E. coliFunction… other model systems include bacteriophage T4 and yeastSummary: What problems do these proteins solve?The ends of (linear) eukaryotic chromosomescannot be replicated by the replisome.Not enoughnucleotides forprimase to startlast laggingstrand fragmentChromosome endsshorten everygeneration!3Telomere shortening signals trouble!1. Telomere shortening releasestelomere binding proteins (TBPs)2. Further shortening affectsexpression of telomere-shortening sensitive genes3. Further shortening leads toDNA damage and mutations.Telomere binding proteins (TBPs)Telomerase replicates the ends (telomeres)Telomere ssDNATelomerase extendsthe leading strand!Synthesis is in the5’-->3’ direction.Telomerase is aribonucleoprotein(RNP). The enzymecontains RNA andproteins.The RNA templatesDNA synthesis. Theproteins include thetelomerase reversetranscriptase TERT.4Telomerase cycles at the telomeresTelomere ssDNATERT proteinTER RNA templateTelomerase extends a chromosome 3’ overhang5Conserved structures in TER and TERTCore secondarystructures shared inciliate andvertebratetelomerase RNAs(TERs). (Sequenceshighly variable.)148-209 nucleotides1000s of nucleotidesTERT proteinsequences conserved1300 nucleotidesStarting and stopping summary1. DNA replication is controlled at the initiation step.2. DNA replication starts at specific sites in E. coli and yeast.3. In E. coli, DnaA recognizes OriC and promotes loading of theDnaB helicase by DnaC (helicase loader)4. DnaA and DnaC reactions are coupled to ATP hydrolysis.5. Bacterial chromosomes are circular, and termination occursopposite OriC.6. In E. coli, the helicase inhibitor protein, Tus, binds 10 terDNA sites to trap the replisome at the end.7. Eukaryotic chromosomes are linear, and the chromosome endscannot be replicated by the replisome.8. Telomerase extends the leading strand at the end.9. Telomerase is a ribonucleoprotein (RNP) with RNA (template)and reverse-transcriptase subunits.6DNA methods summary1. Restriction enzymes cut at specific DNA sites. (N)2. Vectors allow genes to be “cloned” and proteins “expressed”. (N)3. Gel electrophoresis separates DNA on the basis of size.4. DNAs can be synthesized (up to ~100 bases commercially). (N)5. PCR amplifies any target DNA sequence. (N)6. Genes and genomes can be sequenced by chain termination. (N)7. Oligonucleotides can be used to change bases by “site- directedmutagenesis”. (N)8. “Southern” blotting detects sequences by hybridization.9. Microarrays detect gene expression patterns over the genome.10. Genes can be knocked out (deleted) or replaced in prokaryotes andeukaryotes. (N)Restriction enzymes cut DNA at specific sites7Restriction enzymes cut DNA at specific sites• 3 types of ends: 5’ overhang, blunt and 3’ overhang• Cognate methyl transferases protect host genome from digestion.Restriction-modification systems degrade “foreign” DNA.Average frequency of restriction sites in“random” DNA sequencesThe average occurrence of each sequence = 1/4n,where n = the site length and all bases are equally representedSite size468Average frequency 1/2561/4,0961/65,536(1/4 x 1/4 x 1/4 x 1/4)8Lots of different recognition sites knownCore four basesFlanking basesNoneA----TC----GG----CT----AA simple cloning procedure1. Cut “insert” and “vector”DNA with a restrictionenzyme2. Mix and join ends withDNA ligase. The endsshould match forefficient ligation.9 Cloning without DNA ligase1. Prepare open vector and insertwith the same long “sticky” ends + Pol I Klenow fragment + dATP2. Mix and let the ends anneal.3. Transform the nicked plasmid.The plasmid is repaired in vivo.1. Prepare an insert flanked bysites for a site-specific DNArecombinase.2. Mix insert with the closedvector containing therecipient recombination siteand recombinase enzyme. +3. (Have lunch.) Transform.Ligation-independent cloning“Gateway” cloningE +No dT in templateTTTTAATATA“Vectors” allow DNA sequences to be cloned - 1Phage λ for cloningbig (7-25 kb) DNApiecesOri + selectablemarker + cloningsite (polylinker)10“Vectors” allow DNA sequences to be cloned - 2“Reporter” genes:β-gal, GFP . . .Shuttle vectors:move genes betweenorganismsExpression vectors:Make your favorite protein“Vectors” allow DNA sequences to be cloned -3Transient transfection: eukaryotesStable transduction11Gel electrophoresis separates DNA on the basis ofsizeAgarose: big fragments (>300 bp)Acrylamide: smaller fragments, higher resolutionMobility proportional to log MW.Chemical DNA synthesisSequential rounds ofcoupling, oxidationand deprotection ofthe 5’ OH build upthe oligonucleotide.3’5’12Chemical DNA synthesisSequential rounds ofcoupling, oxidationand deprotection ofthe 5’ OH build upthe oligonucleotide.3’5’Frontiers in DNA synthesisCurrently: 100-200 nucleotides routine (Assemble 5 kB)10,000 = largest.Primer set for the human genome (30,000 genes) ~ $104Goal 1: Make yeast chromosome 3: 300 kB without errors! (Jeff Boeke)Goal 2: Assemble 16 X 106 w/o errors for ~$1000 (George Church)13DNA sequencing by partial chain terminationddNTPs terminatethe chainDNA sequencing by partial chain terminationSmall amount of ddGTP + excessdGTP partially terminates chains atCs in the templateddNTPs terminatethe chain14DNA sequencing by partial chain termination1. All fragments start at theprimer2. All fragments ending in aparticular base have adifferent length and adifferent color tag3. Separating the mixture ofproducts by size reveals thesequence.Two strategies for genome sequencingHierarchical Shotgun Sequencing Sequencing15PCR (Polymerase Chain Reaction): isolate andamplify any DNA sequenceN cycles amplifies the target sequence 2N-fold.Copies: 1 2 4 8Site-directed mutagenesis1. Anneal divergentmutagenic primers.2. Replicate entire plasmidwith a DNA pol lacking5’-->3’ exonuclease.3. Select against parentalstrands.16Gene replacement
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