PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21V. cDNA Libraries (converting mRNA into “complementary DNA”Removes the RNA part of RNA:DNA hybridsMBB 407/511Lecture 19:Prokaryotic DNA Replication(Part I)Nov. 11, 2005Landmark Experiments in DNA ReplicationRequirements of ALL DNA PolymerasesMechanism of DNA ReplicationDNA Polymerase I of E. coli and its activitiesI. Why Study DNA Replication?1) To understand cancer—uncontrolled cell division (DNA replication) 2) To understand aging—cells capable of finite # of doublings3) To understand diseases related to DNA repaira) Bloom’s Syndromeb) Xeroderma Pigmentosumc) Werner’s SyndromeKeith Richards (of the Rolling Stones)Example of premature agingNOT caused by a hereditary diseaseII. Landmarks in the Study of DNA ReplicationA. 1953 Watson and Crick:B. 1958 Meselson and StahlFrom the structure of DNA they predicted that the DNA strands could act as templates for the synthesis of new strands: base complementarityThree Potential DNA Replication ModelsNew DNAOld DNAThe Meselson-Stahl Experiment“The most beautiful experiment in biology”Conclusion: DNA is Replicated Semiconservatively:1. The parental strands separate during DNA replication.2. Daughter DNA molecules consist of one new and one old (parental) strand.ParentalIII. General Features of DNA Replication1. require a DNA template and a primer with a 3’ OH end (DNA polymerases can only elongate; no de novo initiation of DNA synthesis) 2. require dNTPs3. synthesize DNA in a 5’ to 3’ direction.All DNA Polymerases:Short RNA primers are needed for initiation in vivo4. require metal ions (Mg2+ or Zn2+) as cofactorsThe Substrates for DNA ReplicationdivalentThe Mechanism of DNA Synthesis121DNA Synthesis Is ExergonicdNTP + (dXMP)n  (dXMP)n+1 + P~P G = -3.5 kcal/moleP~P  2 P  G = -7 kcal/moleTotal: dNTP + (dXMP)n  (dXMP)n+1 + 2 P G = -10.5 kcal/mole2The Role of Metal Ions In DNA SynthesisSteric Constraints Prevent Catalysis of rNTPsReplication of the E. coli Chromosome is BidirectionalReplication of the E. coli Chromosome is SemidiscontinuousReplicates continuously DNA synthesis is going in same direction as replication forkBecause of the anti-parallel structure of the DNA duplex, new DNA must be synthesized in the direction of fork movement in both the 5’ to 3’ and 3’ to 5’ directions overall.Replicates discontinuously DNA synthesis is going in opposite direction as replication forkHowever all known DNA polymerases synthesize DNA in the 5’ to 3’ direction only.The solution is semidiscontinuous DNA replication.Joined by DNA ligase“Now this end is called the thagomizer,after the late Thag Simmons.”IV. DNA Polymerase IThe first DNA polymerase was discovered by Arthur Kornberg in 1957 → DNA Polymerase I ofE. coliA. E. coli DNA Pol I has 3 enzymatic activities:1) 5’ → 3’ DNA polymerase2) 3’ → 5’ exoncuclease (For proofreading)3) 5’ → 3’ DNA exonuclease (For repair: to edit out sections of damaged DNA)1 323Hans Klenow showed that limited proteolysis with either subtilisin or trypsin will cleave Pol Iinto two biologically active fragments.Facts about DNA Synthesis Error Rates:—DNA polymerase inserts one incorrect nucleotide for every 105 nucleotides added.—Proofreading exonucleases decrease the appearance of an incorrect paired base to one in every107 nucleotides added.—Actual error rate observed in a typical cell is one mistake in every 1010 nucleotides added.—Error rate for RNA Polymerase is 1/105 nucleotides.aa 928Klenow Fragment5’ to 3’ Exo.5’ to 3’ Pol & 3’ to 5’ ExoNCKlenow FragmentDNA Repair(Errors fixed after DNA replication)No ProofreadingNick Translation 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’Newly synthesized. DNADNA Polymerase I+5’-dNMPsnicknick5’  3’ exonuclease activity digests DNA5’  3’ polymerase activity replaces the digested DNA with new DNAThey act together to edit out sections of damaged DNAThe 5’ to 3’ Exonuclease and 5’ to 3 Polymerase of Pol I Result in “NickTranslation”: 5’ 3’ 3’ 5’ I 5’ 3’ 3’ 5’5’ → 3’ exonuclease edits damaged DNANewly synthesized. DNADNA Polymerase I+5’-dNMPsnicknickB. ProcessivityDNA Polymerases Can be Processive or DistributiveProcessivity is continuous synthesis by polymerase without dissociation from thetemplate.Processive PolymerizationA DNA polymerase that is Distributive will dissociate from the template after eachnucleotide addition.Distributive Polymerization1 nucleotideUsed inDNAReplicationSuitable forDNA RepairProc.Dist.How to Measure Processivity[32P]-dNTPsssDNAtemplateM13Mg2+5 min. @ 37oCSTOP w/ EDTADNA PolPolyacrylamide GelProcessivity experimentsrequire a large excess oftemplate to Pol toprevent reassociation tothe same template.primer–+Main replicative enzyme Repair enzymeDNA Pol IRNAOkazaki fragment>10 kb1 kbRoles of DNA Pol III and Pol I in E. coliPol III—main DNA replication enzyme. It exists as a dimer to coordinate the synthesis of both the leading and lagging strands at the replication fork. Pol I—repair enzyme to remove RNA primers that initiate DNA synthesis on both strands. It is need predominantly for maturation of Okazaki fragments. 1) Removes RNA primers (5’3’ Exo)2) Replaces the RNA primers with DNA (5’3’ Pol & 3’5’ Exo proofreading)Q: Why do Okazaki fragments initiate with RNA primers?A: Because DNA polymerases require a primer but can’t synthesize them de