Unit 3 Information pathways of gene

1. Unit 3 Information pathways of gene

2. 本章节是考试的重点，尤其是在生化入学试题中所占的比重越来越大，但内容本身难度偏高，所以应做到掌握了解基本概念，基本过程，关键产物和催化酶。不要求对具体内容有详细的了解本章节是考试的重点，尤其是在生化入学试题中所占的比重越来越大，但内容本身难度偏高，所以应做到掌握了解基本概念，基本过程，关键产物和催化酶。不要求对具体内容有详细的了解

3. What’s a Gene? • Such a simple question, such a complicated answer • In part, the answer depends on the what level (molecular, cellular, organismal) we’re interested in • Gene = a sequence of DNA capable of producing some element of biological function

4. replication Reverse tanscription DNA Transcription Ribosome mRNA Translation Polypeptide (protein) The Central Dogma: describes the Flow of Information from DNA  RNA  protein - with the exception of retroviruses: RNA  DNA Cell

5. Chapter 10 DNA biosynthesis ： -Replication （复制） -Reverse transcription （逆转录） Transmission of genetic information on DNA/RNA from parent to daughter

6. DNA REPLICATION = ? • Why? • When? • Where? • Whom? • How?

7. Conservative - Old double stranded DNA serves as a template for two new strands which then join together, giving two old strands together and two new strands together Semi-conservative - Old strands serve as templates for new strands resulting in double stranded DNA made of both old and new strands Old New Old New Old Old Old New + + Old + New Old + New Old + New Old + New Old Dispersive - In which sections of the old strands are dispersed in the new strands + + or THREE HYPOTHESES FOR DNA REPLICATION

8. 1958 – Matthew Meselson and Franklin Stahl 美国科学家马修.梅塞尔(Matthew Meselson)于1930年出生在科罗拉多州的丹佛。他毕业于加州工学院物理化学专业。毕业后留校，1976年到哈佛大学工作 福兰克林.斯塔尔(Franklin Stahl)于1929年出生于马萨诸塞州的波士顿。曾求学于哈佛大学及罗切斯特大学。1955年至1958年在加州工学院工作，其后在密苏里大学工作一年。1970年受聘于俄罗冈大学教授

9. OH NH2 O P HO O N N N N H OH The Meselson-Stahl Experiment • This experiment took advantage of the fact that nucleotide bases contain nitrogen, Thus DNA contains nitrogen • The most common form of Nitrogen is N14with 7 protons and 7 neutrons • N15 is called “heavy nitrogen” as it has 8 neutrons thus increasing its mass by 1 atomic mass unit

10. The Meselson-Stahl experiment

11. Semiconservative replication maintains genetic stability Stability is relative, variation （变异）exists widespreadly in the life world

12. Bidirectionel DNA-Replication

13. 3’ Parental DNA Molecule 5’ Replication Fork 3’ 5’ Replication Forks Replication Forks: hundreds of Y-shaped regions of replicating DNA molecules where new strands are growing

15. Label at both replication forks

16. Bubbles Bubbles Replication Bubbles Replication Bubbles: a.Hundreds of replicating bubbles (Eukaryotes) b.Single replication fork (bacteria)

17. ori ori ori ori bidirectional replication (双向复制) Two replications proceed in opposite directions away from the origin replicon (复制子) DNA region between the neighboring two original site

18. Origin of replication E. coli DNA ter C (termination) Origin is rich in AT bidirectional replication

19. Semi-discontinuous replication • semi-discontinuous • leading strand • lagging strand • Okazaki fragment

20. Okazaki fragment Anti-parallel strands replicated simultaneously -Leading strand is madecontinuouslyin 5’– 3’ -Lagging strand is made discontinuously, in fragments in 5’-3’ C o n t i n u o u s Unzipping S e g m e n t e d Semi-discontinuous replication

21. New strand synthesis always in the 5’-3’ direction? ？

22. The review of DNA replication • 1 strand of DNA must serve as template for synthesis of complementary strand = semi-conservative (1 old and 1 new strand) • Bidirectional and semi-discontinuous • DNA can ONLY be made in a 5' --> 3' direction

23. Enzymes for replication Substrates：dATP，dGTP，dCTP， dTTP. (dNTPs) DNA polymerase（聚合酶）: Template （模板）：single-stranded DNA Primers（引物）：a newly synthesized RNA Other enzymes and factors are also needed

24. 5’- DNA - O - P -N - OH 5’- DNA - OH +dNTPs + P P 1. Chemical reaction of replication DNA Pol I, II and III -the formation of phosphodiester bond A chemical equation: (DNA) n -OH + dNTP → (DNA)n+1 -OH + PPi

25. Substrates: 2. DNA polymerase, DNA-pol: enzymes that make DNA DNA dependent DNA polymerase（DDDP） • A base-paired3’ OH group • A template • dNTPs

26. 1918年3月出生在美国纽约。1941年,23岁获得罗彻斯特大学医学博士学位，1960年和1962年获得法学和科学博士学位 1955年从E.Coli中发现了DNA 聚合酶，为DNA的复制打下了基础。1959年获得诺贝尔奖 Arthur Kornberg 科恩伯格

27. DNA Polymerase Activities • 5’3’ polymerization - enzyme reads template 3'  5’ - dNTPs added to growing molecule at -OH group attached to 3' carbon of (de)oxyribose • 3’5’ exonuclease • 5’3’ exonuclease (Pol I)

28. dNTP DNA pol 5´3´polymerization activity 3 'agctcgatcgtagcctagcgtagcagtgcacgatc 5 ' 5'ucgagcuag-OH 3 ' 3 'agctcgatcgtagcctagcgtagcagtgcacgatc 5 ' 5 'ucgagcuag catcggatcgcatcgtcacgtgctag 3 ' dNTP

29. Endonuclease (内切酶) 5’ 3’ Exonuclease activity 外切酶

30. 5’ 3’ Function of 5´ 3´exonuclease Excision of RNA primer or mutated base pairing

31. 3’5’exonuclease proofreading (Pol I)

32. In prokaryote（原核细胞）： DNA-polⅠ、 Ⅱ、 Ⅲ 、(VI 、V) In eukaryote（真核细胞）： DNA-pol、、、、…

33. Five DNA polymerases in E. coli 1.pol I: (encoded bypolA gene) a) Major repairenzyme for damaged DNA b) Plays secondary role in DNA replication c) Most abundant (400/cell) d) Molecular mass of 103 kD 2. pol II:(encoded bypolBgene) a) Minor DNA repair enzyme b) Molecular mass of 90 kDa

34. Small fragment 323 aa 55aa Hold DNA DNA pol I large fragment （Klenow fragment ,604 aa） 5 ' →3 ' polymerization 3 ' →5 ' exonuclease 5 ' →3 ' exonuclease

35. Five DNA polymerases in E. coli 3.pol III: (encoded bypolC/dnaE gene): a)REPLICASE; de novo synthesis of new strands of DNA b) No 5’ to 3’ exonuclease activity c) There are 10-20/cell d) Many subunits e) Molecular mass of 900 kDa

36.   form the catalytic core  ’           Links two cores    act as a clamp   The model of DNA-pol III

37.           form the catalytic core Links two cores  act as a clamp

38. Five DNA polymerases in E. coli 5.pol V:(encoded byumuD’2C gene) a) SOS repair enzyme of damaged DNA 4.pol IV: (encoded bydinB gene) a) SOS repair enzyme of damaged DNA

39. Polymerase I II III 5’- 3’ Polymerization Yes Yes Yes 3’-5’ Exonuclease Yes Yes Yes 5’-3’ Exonulcease Yes No No Molecules/cell 400 40 20 E. coli DNA Polymerases activity（nt/min） 1000 50100000 Major function Proofreading/ Removal of RNA primers 109,000 Daltons Repair of damaged DNA 120,000 Daltons Replication polymerization 10 subunits 250,000 Daltons Klenow fragment (76,000 Daltons), prepared by mild proteolysis, lacks 5’ to 3’ exonuclease activity and is used in sequencing

40. Eukaryotic DNA polymerases Five identified in mammals (I) (III) (II)   Enzyme (alpha) (delta) (epsilon) (beta) (gamma) Location function Nuclear priming of both strands Nuclear elongationof both strands Nuclear repair &replication Nuclear repair Mitochondrial replication 3’-5’ exonuc. No Yes Yes No Yes 80% 10-15% 2-15% relative activity PRIMASE REPLICASE

41. The fidelity of DNA replication • Base selection: only AT and GC base pairs fit properly in the active site of the polymerase（trans/sis） • The strict base-pairing rule • G-C ； A-T • Proofreading: if a wrong base is inserted, then it is removed and replaced with the correct one before the next one is added DNA synthesis has an extraordinary high fidelity: between 10-8 and 10-10

42. 3. DNA unwinding and its related topology • Helicase（解螺旋酶） • Topoisomerase I、II、Ⅲ（拓扑异构酶） • Single stranded DNA binding protein, SSB （单链DNA结合蛋白）

43. ATP helicase Helicase • Helicase: enzyme which catalyzes the unwinding andseparation(breaking H- Bonds) of the parental double helix dnaA、B、C… DnaA、B、C…

44. DNA Supercoiling

45. topoisomerase,TOPO Topology（拓扑） and topologic isomers DNA positive and negative supercoiling are topologic isomers TOPO Helicase Positive supercoiling （正超螺旋） Normal DNA Negative supercoiling（负超螺旋）

46. TOPOⅠ - not need ATP，cut one strand of the double helix,after the DNA relax,then ligates the nick TOPOⅡ not need ATP when cut the double helix strands ligate the nick,need ATP

47. Helicase The Role of TOPOⅡ（DNA Gyrase）

48. Supercoiled DNA Helicase The Role of TOPOⅡ TOPOⅡ

49. The Role of TOPOⅡ TOPOⅡ

50. The Role of TOPOⅡ TOPOⅡ