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Information Pathways Chapers 24-28 & 9

Explore the components involved in DNA supercoiling and replication processes, their properties, interactive relationships, and how they operate. Discover the structure of chromosomal DNA, the role of topoisomerases, and the steps of DNA replication.

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Information Pathways Chapers 24-28 & 9

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  1. Information PathwaysChapers 24-28 & 9 Nien-tai Hu 胡念台 & Kung-Yao Chang 張功耀 Spring, 2007

  2. Information Pathways Wednesdays : 10:10 ~ 12:00 AM Thursdays : 11:10 ~ 12:00 AM

  3. Biochemistry: 4 ”scenarios”“生化四部曲“ 1. What “components” are involved in a phenomenon or a process? 2. What “biochemical properties” do they possess? 3. What “interactive relationships” exist among these components? 4. How are the “interactive relationships” “translated” into how the components operate in the process?

  4. Chap 24 Chromosomal elements DNA supercoiling The structure of chromosome DNA topoisomerases are the magicians of the DNA world. By allowing DNA strands or double helices to pass through each other, they can solve all of the topological problems of DNA replication, transcription and cellular transactions. James Wang, article in Nature Reviews in Molecular Cell Biology, 2002 Supercoiling, in fact does more for DNA than act as an executive enhancer; it keeps the unruly, spreading DNA inside the cramped confines that the cell has provided for it. Nicholas Cozzarelli, Harvey Lectures, 1993

  5. Do you see E. coli? 1.7 X 103mm v.s. 2 mm Chromosomal DNA bacteria nucleoid

  6. Gene-Protein Co-linearity

  7. Complexity in genomes overview

  8. Complexity in genomes in individual eukaryotic genes

  9. What percentage of human genes encodes RNAs or proteins? repeated sequences

  10. DNA double helix proposed by Watson & Crick 1953 X-ray diffraction pattern of B DNA by Franklin & Wilkins 34 Å/ 10 bp

  11. What do you see? What is supercoiled DNA?

  12. DNA supercoiling is the result of DNA unwinding 84 bp 10.5 bp/turn 12 bp/turn

  13. I. Why supercoiling? II. How is underwound DNA maintained in vivo? • compactness • favorable to strand separation • II. maintained in vivo: • closed circle • or protein-bound • (strands are not free to rotate about each other)

  14. 10.5 bp/turn 2,100 bp Linking number (Lk) Lk is unchanged for INTACT DNA To change the LK, one has to break at least one strand of DNA topoisomer

  15. In vivo, Lk is changed by topoisomerases • Type I topoisomerases single-strand break and rejoining unbroken strand passed through the break Lk changed by increments of 1 • Type II topoisomerases double-strand break and rejoining Lk changed by increments of 2

  16. bacterial type I eukaryotic type IIA E. coli topo I & III (type I) increase Lk topo II & IV (type II) decrease Lk eukaryotes topo I & III (type I) topo IIa and IIb (type II) can not introduce negative supercoiling

  17. DNA unwinding enhances its compaction two forms of negative supercoiling stable in solution; but extended and branched Lest stable (stablized by bound-protein) but more compact

  18. molecular structure of nucleosome 200 bp DNA/ 146 bp around histone core

  19. nearly identical in all eukaryotes Modifications of histones: methylation (CH3-), acetylation (CH3COO-), phosphorylation, etc.

  20. beads on a string electron micrograph of nucleosome

  21. Hierarchical structure of Chromosome chromatin -coil -rosette -(nuclear scaffold with 6 loops) -30-nm fiber -nucleosome (11 nm diameter) 10,000-fold 100-fold 7-fold compaction

  22. You must recognize this

  23. Chap 25 DNA Replication DNA Repair DNA Recombination I have never known a dull enzyme. Arthur Kornberg, in the essay “For Love of Enzymes”, 1975

  24. DNA replication Is DNA replicated the way as proposed by Watson & Crick?

  25. Characteristics of DNA Replication 1. Semi-conservative v.s. conservative or dispersive 2. Bidirectional v.s. unidirectional

  26. The Meselson-Stahl Experiment (1957) results observation grown in 15N-containing medium for many generations interpretation 解讀 grown in 14N-containing medium for 1 generation Is this the only interpretation ?Are there other possible interpretations? Is there way to rule them out? grown in 14N-containing medium for the 2nd generation

  27. Steps of DNA replication • Initiation: where and how replication fork(s) is (are) generated? • Elongation: how does replication fork move? • Termination

  28. How is DNA replication initiated? E. coli oriC (chromosomal origin of replication)

  29. Cell cycle-regulated replication initiation E. coli oriC replicators ARS DnaA ORC HU cyclin-CDK CDC6/CDT1 DnaC DnaB MCM2~7 Science (1996) 274:1659 ARS, autonomously replicating sequences ORC, origin recognition complex CDC, cell division cycle-related proteins MCM, minichromosome maintenance proteins CDK, cyclin-dependent kinases

  30. What is the chemistry of DNA synthesis in the cell?

  31. Replication Elongation at Replication Fork template newly synthesized

  32. Events at Replication Fork 1. Unwinding 2. Negative supercoiling 3. Polymerization: 5’ -> 3’ synthesis leading strand: synthesized continuously lagging strand: synthesized discontinuously as Okazaki fragments evidences: biochemical & EM

  33. Enzymes involved in the events at Replication fork 1. Helicase (DnaB) 2. DNA gyrase (topoisomerase II) 3. DNA polymerase III (DnaE): multi-subunit core enzyme: a, e, q subunits 4. Single-stranded DNA binding protein (SSB) 5. Primase (DnaG) 6. DNA polymerase I (PolA) 7. DNA ligase

  34. DNA gyrase functions to remove positive supercoils that normally form ahead of the growing replication fork MCB Figure 12-18

  35. DNA polymerase I of E. coliisolated by A. Kornberg and colleagues, 1957 1. structure: digested by protease into a large fragment (Klenow fragment) and a small fragment 2. 3 enzyme activities from 1 single polypeptide: 1. 5’->3’ polymerization (Klenow fragment) cannot initiate DNA synthesis de novo, requires a primer 2. 3’->5’ exonuclease (Klenow fragment) proof-reading 3. 5’->3’ exonuclease (small fragment) role in DNA replication--removal of RNA primer role in DNA repair (excision repair)

  36. Klenow fragment from a thermophilic bacterium Bacillus stearothermophilus E. coli template Stryer PDB ID: 3BDP

  37. Properties of E. coli DNA Polymerase I & III

  38. How does 3’-exonuclease activity help proofreading? What is the relationship between proofreading and mutation? rate of making mistakes in replication: without proofreading: 10-4~10-5 bp with proofreading: 10-6~10-8 bp

  39. Why is 5’-exonuclease activity of Pol I “essential”? Nick “translation”

  40. What makes Pol III synthesize at faster rate and more “processive” than Pol I? a donut-shaped, dimeric protein complex b clamp

  41. a, alpha; b, beta; e, epsilon; q, theta; g, gamma d, delta; c, chi; , tsai; t, tau

  42. DNA polymerase III of E. coli 1. lacks 5’->3’ exonuclease activity 2. holoenzyme components: (core: a e q) DNA polymerase III* : (a eq)2 / g / t2 b2 : sliding clamp g complex (g, d, d’, c , y) as clamp loader 3. Mutation in DNA polymerase III is lethal. 4. 3’-exonuclease associated with e (DnaQ)

  43. How does DNA ligase seal the nick?

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