Handout for Lecture 19: Prokaryotic DNA Replication I (Nov. 11, 2005)OutlineIII. General Features of DNA ReplicationMOLECULAR BIOLOGY AND BIOCHEMISTRY 694:407 & 115:511Handout for Lecture 19: Prokaryotic DNA Replication I (Nov. 11, 2005)Dr. Marty NemerofWaksman 19(732) [email protected]“When I was a boy of fourteen my father was so ignorant I could hardly stand to be around the old man. By the time I was twenty-one, I was astonished at much he had learned in only 7 years!”—Mark TwainOutlineI. Why Study DNA Replication?II. Landmarks in the Study of DNA ReplicationA. 1953—Watson and CrickB. 1958—Meselson and StahlII. General Features of DNA ReplicationA. Rules for all DNA Polymerases1. require a template and a primer with a 3’ OH end (no de novo initiation)2. synthesize in a 5’ to 3’ direction 3. require dNTPs4. require metal ions (Mg2+ or Zn2+) as cofactorsB. Mechanism of DNA ReplicationC. Replication is BidirectionalD. Replication is SemidiscontinuousIV. DNA Polymerases of E. coliA. DNA polymerase I1. 5’ to 3’ polymerase (DNA synthesis activity)2. 3’ to 5’ exonuclease (proofreading activity)3. 5’ to 3’ exonuclease (editing)B. ProcessivityV. Reading listA. Chapter 28 of Biochemistry, Garrett and Grisham, 3rd EditionB. Chapter 8 of Molecular Biology of the Gene, Watson et al, 5th Edition1VI. What you need to know for Exam #3I. Why Study DNA Replication?1) To understand cancer-- the uncontrolled cell division (uncontrolled DNA replication).2) To understand aging--cells are capable of a finite number of doublings.3) To understand diseases associated with defects in DNA repair.1) CancerCells are carefully controlled in the number of cell doublings that they are capable of as well as when cell division will occur. In cancer the control of initiation of replication is lost2) AgingFor example, fibroblast cells (connective tissue) in culture will double for about 50 generations. Then they enter “senescence.” Senescent cells are no longer capable of dividing yet remain metabolically active. In addition, they exhibit changes in form and function, which may lead to age-related changes such as the diference between the supple skin of a child and the wrinkled skin of the elderly. 3) DNA repair diseasesThere are several diseases that cause premature aging or sensitivity to UV light.Examples include:a) Bloom’s Syndrome, a cancer-prone genetic disorder due to genetic instability in the form of increasedfrequencies of breaks of the chromosomes.b) Xeroderma Pigmentosum, a human DNA repair deficiency syndrome leading to predisposition to sun-light-induced skin cancer.c) Werner’s Syndrome, a premature aging disease that begins in adolescence or early adulthood and results in the appearance of old age by 30-40 years of age. 2II. Landmarks in the Study of DNA ReplicationA. 1953 Watson and CrickFrom the structure of DNA they predicted that the DNA strands could act as templates for synthesis of new strands:B. 1958 Meselson and StahlThree potential DNA replication models and their predicted outcomes3The Meselson and Stahl Experiment4Fig. 28.3, G&G 3rd ed.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.III. General Features of DNA ReplicationA. Rules for All DNA Polymerases:1. They require a primer annealed to a template. The primer must have a free 3’ OH to which nucleotides are added (DNA polymerases can only elongate; no de novo initiation of DNA synthesis).2. They require dNTPs.3. They synthesize DNA in a 5’ to 3’ direction.4. They require metal ions such as Mg2+ or Zn2+ as cofactors.The Substrates for DNA Replication5Fig. 2-9, Watson 5th ed.B. Mechanism of DNA ReplicationThe Mechanism of DNA SynthesisDNA Synthesis is an Exergonic Reaction6Fig. 8-1, Watson, 5th ed.Fig. 8-2, Watson, 5th ed.The Role of Metal Ions In DNA Synthesis7Fig. 8-6, Watson, 5th ed.Steric Constraints Prevent Catalysis of rNTPs by DNA PolymerseFig. 8-4, Watson, 5th ed.8Because 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.However all known DNA polymerases synthesize DNA in the 5’ to 3’ direction only.9Fig. 28.5, G&G 3rd ed.B. Replication is BidirectionalC. Replication is SemidiscontinuousFig. 28.6, G&G 3rd ed.The solution is semidiscontinuous DNA replication.Leading Strand-replicates continuouslyLagging Strand-replicates discontinuously -consists of Okazaki Fragments (single-stranded DNA chains 1000-2000 nucleotides long, which need tobe joined by DNA ligase.-the parental strand forms a “trombone structure”IV. DNA Polymerase IThe first DNA polymerase was discovered by Arthur Kornberg in 1957  DNA Polymerase I of E. 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)1 323 Hans Klenow showed that limited proteolysis with either subtilisin or trypsin will cleave Pol I into two biologically active fragments.Facts about DNA Synthesis Error Rates:—DNA polymerase inserts one incorrect nucleotide for every 105 nucleotides added.10aa 928Klenow Fragment5’ to 3’ Exo. 5’ to 3’ Pol & 3’ to 5’ ExoN C—Proofreading exonucleases decrease the appearance of an incorrect paired base to one in every 107 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.The 5’ to 3’ Pol and 3’ to 5’ Exo Activities Have Distinct Active Sites11The 3’ to 5’ Exonuclease (Proofreading) Activity of DNA Pol I3) 5’ to 3’ Exonuclease of DNA Pol IDNA Pol I Will Perform “Nick Translation” on a Nicked dsDNA Template 5’ 3’ 3’ 5’ 12Newly synthesized. DNADNA Polymerase InickFig. 28.8, G&G 3rd ed.5’ 3’ 3’ 5’5’  3’ exonuclease activity digests DNA5’  3’ polymerase activity replaces the digested DNA with new DNAB. ProcessivityDNA Polymerases Can be Processive or DistributiveProcessivity is continuous synthesis by polymerase without dissociation from the template.13+5’-dNMPsnickProcessive PolymerizationA DNA polymerase that is