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Rutgers University MBB 408 - LECTURE NOTES

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CoreCoreLaggingHandout for Lecture 20: Prokaryotic and Eukaryotic DNA Replication (Nov. 15, 2005)OutlineEukaryotic DNA ReplicationMOLECULAR BIOLOGY AND BIOCHEMISTRY 694:407 & 115:511Handout for Lecture 20: Prokaryotic and Eukaryotic DNA Replication (Nov. 15, 2005)Dr. Marty NemerofWaksman 19(732) [email protected]“I always wanted to be somebody, but I see now I should have been more specific!”—Lily Tomlin, stand-up comic.OutlineProkaryotic DNA Replication (cont.)IV. DNA Polymerases of E. coliC. DNA Polymerase III1. the -sliding clamp2. the clamp loader3. the asymmetric dimerD. Replication Fork ProteinsE. Synthesis of Okazaki fragmentsF. Maturation of Okazaki fragmentsEukaryotic DNA ReplicationI. Eukaryotic DNA PolymerasesA. DNA Polymerase B. DNA Polymerase II. Additional Eukaryotic Replication ProteinsA. PCNAB. RPAC. RFCIII. Leading and Lagging Strand SynthesisA. Polymerase SwitchingB. Maturation of Okazaki FragmentsC. Multiple Origins of ReplicationIV. TelomeresA. Linear Ends of ChromosomesB. Telomerase1. How Telomerase Adds Telomeric Repeats.2. How Telomeres Protect the Linear Ends of ChromosomesV. What you need to know for Exam #41Reading ListA. Biochemistry, Garrett and Grisham,1. Chapter 28 (3rd ed.) or Chapter 30 (2nd ed.)A. Molecular Biology of the Gene, Watson et al., 5th Edition1. Chapter 8—The Replication of DNADNA Polymerase III Holoenzyme has 10 SubunitsPol III Core- complex of subunits that catalyzes DNA synthesis subunit- has polymerase activity2 subunitcore complex = “clamp loader”(Fig. 28.10, G&G 3rd ed.) subunit- has the 3’-5’ exonuclease activity (proofreading) Subunit (sliding clamp)- a  subunit dimer tethers the “core” polymerase to the template and is responsible for the high processivity of the holoenzyme. Complex (clamp loader)- transfers the sliding clamp to each strand of the DNA template. It can also unclamp the 2 sliding clamp from the DNA template when needed.If the clamp loader releases the 2 sliding clamp from the DNA template the polymerase will dissociate from the template.The  -Subunit Dimer “Clamp” Increases the Processivity of DNA Polymerase III3(Fig. 8.17, Watson 5th ed.)Pol III is an Asymmetric Dimer4Asymmetric Dimer: the dimeric DNA Polymerase is asymmetric. The -complex (clamp loader) is present on only one half of the dimer because it is needed predominantly to copy the lagging strand. This enables the asymmetric dimer to periodically dissociate from the lagging strand template in order to start a new Okazaki fragment.5Core CoreSynthesizestheLeadingStrandSynthesizestheLaggingStrandAdditional Replication Fork ProteinsHelicase- a 5’-3’ helicase that also unwinds the DNA double helix. It is in a complex with Primase.Primase- synthesizes RNA primers needed to initiate DNA synthesis on both strands (greatest need is for Okazaki fragments).SSB- Single Stranded Binding Protein that coats the ssDNA template strands ahead of thepolymerase to maintain them as single-stranded. 1) Exists as a tetramer of 19 Kdal monomers2) Unfolds secondary structure ahead of the moving polymerase3) Protects against nucleases4) Stimulates Pol III synthesis5) Is displaced by the moving polymerase6) Is recycled at the replication forkDNA Gyrase- a topoisomerase that relaxes the DNA double helix that can become twisted due to the unwinding of DNA at replication fork.Synthesis of Okazaki Fragments by the Pol III Holoenzyme6At each replication fork is the Replisome, a multi-protein replication machine that consists of:1) DNA unwinding proteins2) the priming apparatus (Primosome)3) DNA Pol III holoenzyme7When an Okazaki fragment reaches the primer of the previous Okazaki fragment, that is a signal for the clamp loader to unclamp the 2 sliding clamp from the DNA template. As a result, the Pol III holoenzyme dimer detaches from the laggingThe clamp loader will reclamp the 2 sliding clamp to the template where the next primer is encountered.Maturation of Okazaki Fragments by Pol I(1) The 5’-3’ Exonuclease activity of DNA Pol I progressively chews away the RNA primer on Okazaki fragments. (2) The 5’-3’ Pol and 3’-5’ Exo activities of Pol I replace the degraded RNA primer with DNA.(3) The nick is sealed by DNA Ligase (only ligates DNA to DNA) What happens at the junctions between Okazaki fragments?3’--ApTpCpApGpTpApTpCpApUpGpCpUpUpApU CpGpTpApCpGpApApTpGpTpC------5’5’--TpApGpTpCpApTpApGpTpApCpGpApApTpApGpCpApTpGpCpTpTpApCpApG------3’3’--ApTpCpApGpTpApTpCpApUpGpCpUpUpA TpCpGpTpApCpGpApApTpGpTpC------5’5’--TpApGpTpCpApTpApGpTpApCpGpApApTpApGpCpApTpGpCpTpTpApCpApG------3’3’--ApTpCpApGpTpApTpCpApUpGpCpUpU ApTpCpGpTpApCpGpApApTpGpTpC------5’5’--TpApGpTpCpApTpApGpTpApCpGpApApTpApGpCpApTpGpCpTpTpApCpApG------3’3’--ApTpCpApGpTpApT CpApTpGpCpTpTpApTpCpGpTpApCpGpApApTpGpTpC------5’85’P3’OHDNA Pol I+DNA Ligase3’5’3’5’Lagging strandtemplate5’ 3’3’5’Reminder: Pol I also removes RNA primers on the leading strand.OkazakifragmentRNARNADNARNARNA5’P3’OH5’--TpApGpTpCpApTpApGpTpApCpGpApApTpApGpCpApTpGpCpTpTpApCpApG------3’3’--ApTpCpApGpTpApTpCpApTpGpCpTpTpApTpCpGpTpApCpGpApApTpGpTpC------5’5’--TpApGpTpCpApTpApGpTpApCpGpApApTpApGpCpApTpGpCpTpTpApCpApG------3’I. Eukaryotic DNA PolymerasesDNA Polymerase  DNA Polymerase -involved in initiation -the DNA-replicating enzyme-synthesizes an RNA primer then adds dNTPs of mitochondria-a complex of four subunits-50-kD and 60-kD are primase subunits;180-kD subunit DNA polymerase-synthesizes 8-10 nt RNA primers, then adds DNA to the RNA primers-low processivity of DNA synthesis (200 nt)-has no 3’ -5’ exonuclease activity (proofreading), yet has high fidelityDNA Polymerase -the principal DNA polymerase in eukaryotic DNA replication -has 3’-5’ exonuclease activity -consists of a 125 kdal and a ~50 kdal subunit -the 50 kd subunit interacts with PCNA (Proliferating Cell Nuclear Antigen) = sliding clamp-is highly processive when in association with PCNADNA Polymerase 9-required for replication, but its role is unclear-may substitute for DNA polymerase  in lagging strand synthesisDNA Polymerase -role in DNA repair (doesn’t participate in replication)II. Additional Proteins Involved in Eukaryotic DNA SynthesisPCNA (Proliferating Cell Nuclear Antigen)-confers high processivity to DNA Polymerase -the eukaryotic


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