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DNA Replication , DNA Damage and Repair

DNA Replication , DNA Damage and Repair. Outline. Central Dogma( 中心法则 ) DNA Replication( 复制 ) Testing Models for DNA replication Semi-conservative replication ( 半保留复制 ) Evidence for Semi-Discontinuous ( 半不连续 ) Replication E. coli DNA polymerases Details of DNA Replication Three steps

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DNA Replication , DNA Damage and Repair

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  1. DNA Replication , DNA Damage and Repair

  2. Outline • Central Dogma(中心法则) • DNA Replication(复制) • Testing Models for DNA replication • Semi-conservative replication(半保留复制) • Evidence for Semi-Discontinuous(半不连续) Replication • E. coli DNA polymerases • Details of DNA Replication Three steps • 1) Initiation（起始） • 2) Elongation（延伸） • 3) Termination and Separation • （终止与分离） • Eukaryotic DNA ReplicationLike E. coli, but more complex • DNA Damage and Repair • Types of Repair

  3. Central Dogma Central Dogma 中心法则

  4. Central Dogma DNA→RNA→Protein

  5. Central Dogma Central Dogma Gene expression Transcription Translation Retro-transcription Replication Replication

  6. Central Dogma 转录（transcription）: The process in which DNA is used to synthesize RNA is called transcription. 翻译（translation): Protein synthesis is referred to as translation. 基因表达（gene expression): Gene expression is the process by which a gene's information is converted into the structures and functions of a cell.

  7. 序 论 转录（transcription）: DNA把基因信息传递给RNA的过程。 翻译（translation): 是以mRNA为模板,按照三个核苷酸决定一个氨基酸的原则,把mRNA上的遗传信息转换成蛋白质分子中特定的氨基酸序列的过程。 基因表达（gene expression): 通过转录和翻译，用基因的遗传信息在细胞合成了有功能意义的各种蛋白质。

  8. Central Dogma Coding strand, Sense strand, Crick strand Template strand, antisense strand, Watson strand Transcription Translation

  9. Happy Birthday,Double Helix

  10. DNA Replication

  11. DNA Replication

  12. DNA Replication In 1953, James Watson and Francis Crick deduced the Secondary Structure of DNA Double-helix Structural Model (双螺旋结构模型). This was one of the most important biological advances, since it led to an understanding of the relationship of the DNA structure to its function, particularly to the way it was replicated (复制).

  13. DNA Replication "Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid"(Nature, April 25, 1953. volume 171:737-738.) • "...It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material. The structure itself suggested that each strand could separate and act as a template for a new strand, therefore doubling the amount of DNA, yet keeping the genetic information, in the form of the original sequence, intact. "

  14. DNA Replication • Can you remember The Basic Features of B-DNA?

  15. DNA Replication • Can you remember The Basic Features of B-DNA? • There have 5 features and 6 parameters.

  16. The Basic Features of B-DNA 4. Bases are Restricted Pairing on the Inside A purine-pyrimidine pair fits PERFECTLY. Three hydrogen bonds （氢键）hold the deoxyguanosine nucleotide to the deoxycytidine nucleotide, whereas the other pair, the A-T pair, is held together by two hydrogen bonds.

  17. The Basic Features of B-DNA 5. A Pair of Chains are Complementary Sequences The bases are complementary, with A on one side of the molecule you only get T on the other side, similarly with G and C. If we know the base sequence of one strand we know its complement.

  18. DNA Replication DNA Replication General Features

  19. DNA Replication • DNA Replication General Features 1) Many enzymes and proteins are required 2) Template & dNTPs/Mg 2+ are required 3) Semi-conservative (半保留） A key experiment designed by M. Meselson and W. F. Stahl （1958） 4) DNA Unwinding is necessary 5) A Primer (引物） with a free 3' -OH group is required

  20. DNA Replication DNA ReplicationGeneral Features 6) Only in the 5′→3′direction 7) Specific Origin of Replication-Ori C(大肠杆菌的复制原点) 8) Bi-directional (With some exceptions) 9) Semi-discontinuous (半不连续) Replication fork (复制叉) ， Leading strand (前导链), Lagging strand (后随链)and Okazaki fragments (冈崎片段) 10) Highly processive (进行性), Highly ordered and Extremely accurate

  21. DNA Replication Testing Models for DNA replication Matthew Meselson and Franklin Stahl (1958)

  22. DNA Replication Matthew Meselson and Franklin Stahl more recently Faculty member at Harvard Mechanisms of Molecular Evolution Faculty Chair for CBW Studies Faculty member at U. of Oregon Meiotic Recombination

  23. DNA Replication 1958年Meselson和Stahl用同位素示踪和密度梯度离心的方法证明了DNA的半保留复制

  24. DNA Replication Testing Models for DNA replication Meselson and Stahl (1958) Bacterial culture Grow for several generations Bacterial culture withdense DNA 15NH4Cl (Sole N source) This is the starting material for the experiment Density labeling experiment on E. coli (bacterial) DNA

  25. Harvest cells and resuspend in media with 14NH4Cl as the sole N source 14NH4Cl Grow for 1 generation Grow for another generation Grow for another generation etc Bacterial culture withdense DNA Bacterial culture “0 generation” Meselson and Stahl (continued) Harvest some cells “2nd generation” Harvest some cells “1st generation” For each generation isolate the DNA and spin through a density (CsCl) gradient). Detect DNA in the gradient (by UV absorption) Monitor how many DNA bands there are after each generation

  26. DNA Replication

  27. DNA Replication Meselson and Stahl Original Data

  28. DNA Replication Semi-conservative replication?半保留复制的定义

  29. DNA Replication Semi-conservative replication: After the two strands separate, each serves as a template for the synthesis of a complementary strand. (In other words, each of the two new DNA molecules contains one old strand and one new strand.) This process, referred to as semiconservative replication.

  30. DNA Replication • Since DNA replication is semiconservative, therefore the helix must be unwound.

  31. DNA Replication • John Cairns (1963) showed that initial unwinding is localized to a region of the bacterial circular genome, called an “origin” or “ori” for short.

  32. DNA Replication Bacterial culture *T *T *T *T Grow cells for several generations Small amounts of 3H thymidine are incorporated into new DNA in media with low concentration of 3H- thymidine *T *T *T Add a high concentration of 3H- thymidine Grow for brief period of time *T *T *T *T *T Dense label at the replication fork where new DNA is being made All DNA is lightly labeled with radioactivity *T *T *T *T *T *T *T *T *T *T *T *T *T *T *T *T *T *T *T *T *T *T John Cairns Cairns then isolated the chromosomes by lysing the cells very very gently and placed them on an electron micrograph (EM) grid which he exposed to X-ray film for two months.

  33. DNA Replication Label at both replication forks Evidence points to bidirectional replication

  34. DNA Replication

  35. DNA Replication

  36. DNA Replication 3’ 5’ Primer 3’ 5’ Direction of unwinding 3’ Primer Primer 5’ 5’ 3’ 3’ 5’ DNA Replication is Semi-discontinuous Consider one replication fork: Continuous replication Discontinuous replication

  37. DNA Replication 在复制的起始点处，DNA双链部分解开为单链，形成叉子形状称复制叉（replication fork）。

  38. DNA Replication Evidence for the Semi-Discontinuous replication model was provided by the Okazakis (1968)

  39. DNA Replication Flood with non-radioactive T Add 3H Thymidine Bacterial culture Allow replication To continue For a SHORT time (i.e. seconds) Bacteria are replicating smallest largest Evidence for Semi-Discontinuous Replication (pulse-chase experiment) Harvest the bacteria at different times after the chase Isolate their DNA Separate the strands (using alkali conditions) Run on a sizing gradient Radioactivity will only be in the DNA that was made during the pulse

  40. DNA Replication Pulse Chase 3’ 5’ Primer 3’ * * * * * * smallest 5’ Direction of unwinding Primer 3’ 5’ 3’ largest Primer 5’ 3’ 5’ Results of pulse-chase experiment

  41. DNA Replication DNA replication is semi-discontinuous Continuous synthesis Discontinuous synthesis

  42. DNA Replication Many enzymes and proteins are required in DNA Replication.

  43. DNA Replication Many enzymes and proteins are required in DNA Replication. Do you know how many enzymes are required in DNA Replication?

  44. DNA Replication Enzymes and Proteins Involved in DNA Replication • Topoisomerase（拓扑异构酶） • DNA Helicase（DNA解链酶） • Single-stranded DNA binding proteins（ SSB，单链结合蛋白） • Primase（引发酶）- formation of RNA primers • DNA –dependent DNA polymerase III (DNA pol, DNA聚合酶III） • The Enzymes responsible for removing RNA primers (DNA polymerase I) •   DNA ligase （DNA 连接酶）-joining of Okazaki fragments

  45. DNA Replication

  46. DNA Replication

  47. DNA Replication E. coli DNA polymerases • Identification Kornberg and DNA pol I (Kornberg enzyme) • Structure and Function of DNA pol I A multi-functional enzyme • DNA pol III is a major polymerase involved in E. coli chromosome DNA replication

  48. DNA Replication Currently a faculty member at Stanford School of Medicine DNA polymerase I（pol I）：1956年Kornberg等在Ecoli中发现的第一个DNA聚合酶，是DNA复制研究中的重要里程碑，因此于1959年Kornberg获得诺贝尔化学奖。

  49. DNA Replication DNA Pol I from E. coli is 928 aa (109 kD) monomer A single polypeptide with at least three different Enzymatic activities! • How Amazing!!! • a 3’ to 5’ exonuclease activity • a 5’ to 3’ exonuclease activity • a 5’ to 3’ DNA polymerizing activity

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