DNA replication eukaryotes Class 4 .pdf

DNA Replication in Eukaryotes Semi-conservative replication of DNA DNA Replication: Prokaryotes versus Eukaryotes Prokaryotes Eukaryotes ________________________________________________ § Occurs in cytoplasm § One origin/DNA § Ori: ~245 nucleotides § Two forks/DNA § one replicon § Initiation by...

DNA Replication in Eukaryotes Semi-conservative replication of DNA DNA Replication: Prokaryotes versus Eukaryotes Prokaryotes Eukaryotes ________________________________________________ § Occurs in cytoplasm § One origin/DNA § Ori: ~245 nucleotides § Two forks/DNA § one replicon § Initiation by Dna A and Dna B § DNA gyrase § Okazaki frag.: 1000-2000 bases § Very rapid 2000 bp/sec Occurs inside nucleus Multiple origin of replication Ori: 150 nucleotides Multiple forks/DNA 50,000 replicon initiates by Origin Recognition Complex (ORC) Topoisomerases Okazaki frag.: 100-200 bases Slow 100 bp/sec Replication Bubble at the Origin of Replication Figure 5-23 Molecular Biology of the Cell Eucaryotic chromosomes have multiple origins of replication Yeast chromosome III 180 genes 19 replication origins Figure 5-30 Molecular Biology of the Cell DNA replication takes place only during S-phase Figure 5-29 Molecular Biology of the Cell Initiation of DNA replication in eucaryotes During each cell cycle all the DNA must be copied once and only once Figure 5-31 Molecular Biology of the Cell Regulation of replication intitiation in procaryotes Figure 5-26 Molecular Biology of the Cell Eukaryotic DNA polymerases Polymerase alpha (Pol α): Major polymerase for DNA replication, consists of 4 subunits; 2 α and 2 primase Polymerase beta (Pol b): involved in DNA repair Polymerase gamma (Pol g): Mitochondrial DNA replication DNA synthesis catalyzed by DNA polymerases Figure 5-4 Molecular Biology of the Cell Editing by DNA polymerase Figure 5-9 Molecular Biology of the Cell Tautomeric form of bases Base miss-pairing Proofreading Function of DNA Polymerase Figure 5-8 Molecular Biology of the Cell Synthesis of the Lagging Strand Figure 5-11 Molecular Biology of the Cell Function of DNA Ligase Figure 5-12 Molecular Biology of the Cell DNA Helicase Structure Figure 5-13, 5-14 Molecular Biology of the Cell Single Strand DNA Binding Protein Figure 5-15 Molecular Biology of the Cell (© Garland Science 2008) Regulated Sliding Clamp of DNA Polymerase Figure 5-17 Molecular Biology of the Cell Co-operative Action of Different Replication Proteins Figure 5-18 Molecular Biology of the Cell (© Garland Science 2008) Problem Winding Problem that Arises as the Replication Fork Proceeds Figure 5-20 Molecular Biology of the Cell Topoisomerase I Figure 5-21 Molecular Biology of the Cell Topoisomerase II Action of Topoisomerase II Figure 5-22 Molecular Biology of the Cell Topoisomerase Inhibitors • Topoisomerase inhibitors are used for cancer therapy. • Irinitecan and Topotecan are topoisomerase I inhibitors, used to treat colorectal cancer and ovarian cancer, respectively • Doxorubicin (Adriamycin), Etoposide (Etopophos), and ellipticine are in clinical use for different types of cancer • Alll of these anti-cancer agents increase DNA damage in the targeted rapidly growing tumor cell, however non-cancerous tissues can also be affected, leading to more general toxicity and unpleasant side effects. Formation of nucleosome behind a replication fork Figure 5-32 Molecular Biology of the Cell Telomere Telomeric caps Telomere Loops Shortening of telomere length Telomerase Figure 5-33 Molecular Biology of the Cell Telomere Replication Figure 5-41 Molecular Biology of the Cell (© Garland Science 2008) T-Loop at the Telomere Figure 5-35 Molecular Biology of the Cell Problem Diseases caused by defects in DNA Replication and changes in chromatin structure • Warner Syndrome: Dramatic and rapid appearance of features associated with aging, typical onset after puberty, live into their late forties or early fifties. Defective WRN gene (RecA helicase-like). Mutation caused a shorter protein, which degrades rapidly. • Friedreich’s Ataxia: Progressive damage to the nervous system, poor coordination, Vision and hearing impairment, slurred speech, heart disease,diabetes etc. Caused due to GAA repeat introduced into frataxin gene intron that leads to heterochromatin formation and gene silencing. Problem If replication had to be accomplished in an 8-hour S phase and replication forks moved at 50 nucleotides per second, what would be the minimum number of origins required to replicate the human genome? (Human genome comprises a total of 6.4 X 109 nucleotides on 46 chromosomes) Assuming that there would be no time constrains on replication of the complete genome, what would be the minimum number of origins that would be required? Problems Q. 1. What would you expect if dideoxycytidine triphosphate (ddCTP) were added to a DNA replication reaction in large excess over the concentration of the deoxocytidine triphosphate (dCTP)? Q. 2. Predict which of the following proteins, if temperature sensitive would display a quick-stop phenotypes; DNA topoisomerase I, DNA helicase, DNA primase, and DNA ligase Class Objectives Sep 3: DNA Replication (Eukaryotes) After this class students will learn, uthe mechanism of DNA replication in eukaryotes and what are the differences with prokaryotic DNA replication uthe requirements for multiple replication origin in eukaryotes uRe-assembly of nucleosomes after replication uTopoisomerases I and II, How they work? What are the differences? ulthe order of action of different replication enzymes; such as helicase, primase, ligase, DNA polymerase, and topoisomerase uthe role of the proof-reading function of DNA polymerase uTelomere cap and telomerase uHow telomerase prevents chromosome end shortening