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Biochemistry

Biochemistry. Dept. of Biochemistry and Molecular Biology. Professor Wu Yaosheng 2009-10. 或許這就是一條普通的路吧， 楓葉給了它讓人感動的理由。. 心灵的家，平静的地方。 远方寺庙里传来悠悠的吟唱。. 3. Expression and transmission of genetic information.

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Biochemistry

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  1. Biochemistry Dept. of Biochemistry and Molecular Biology Professor Wu Yaosheng 2009-10

  2. 或許這就是一條普通的路吧， 楓葉給了它讓人感動的理由。 心灵的家，平静的地方。 远方寺庙里传来悠悠的吟唱。

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  4. Expression and transmission of genetic information The delivery of genetic signals The central dogma of molecular biology 4

  5. Several important concepts 1. Gene——It is a functional segment on DNA which can code a product with biological activity, including a RNA or a protein. 2. The molecular biological central dogma and its complement RNA replication replication translation transcription RNA protein DNA reverse transcription 3.Gene expression 5

  6. Chapter 10 DNA Biosynthesis

  7. G C C G C G G C A T T A T A G C C G C G G C A T T A T A G C C G C G G C A T T A T A The replication of DNA double helix G C C G A T T C G G C T A A 7

  8. 3’ P P P P P P C G template G C A A A T T A T P P P P P P OH T 5’ Mechanism of DNA replication 5’ The direction of new strand 3’ 8

  9. Main Contents ★ General Features of DNA Replication ★ Eukaryotic DNA Replication ★ Prodaryotic DNA Replication ★ Other Modes of DNA Replication ★ DNA Damage and Repair 9

  10. Key Points ★ Major Features of DNA Replication ★ System of DNA Replication ★ The differences of DNA replication between in eukaryote and in prokaryote ★ DNA polymerases in prokaryote and in eukaryote ★ DNA telomere and telomerase 10

  11. Section One General Features of DNA Replication

  12. The characteristics of DNA replication • Semi-conservatively replication • Semi-discontinuously replication • Bi-directionally replication • High fidelity • Primer needed 12

  13. DNA Semiconservative Replication Bacteria grow in medium contained 15NH4CI as the sole nitrogen source. Density gradient centrifugation 15N- parent DNA (heavy isotope of nitrogen) medium contained 14NH4CI The first generation medium contained 14NH4CI The second generation 13

  14. Semi-conservatively replication During the process of DNA replication, both strands of the parental DNA serve as template for the synthesis of two new double-stranded DNA molecules each of which has one original template strand and one strand of newly synthesized DNA. Thus, replication is Semi-conservative 14

  15. G C C G C G G C A T T A T A G C C G C G G C A T T A T A G C C G C G G C A T T A T A DNA replication G C C G A T T C G G C T A A Semiconservative replication 15

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  17. Semi-discontinuously replication DNA synthesis proceeds in a 5’→3’ direction on each strand of the parental DNA. On the strand with fork orientation (the leading strand), the new DNA is synthesizedcontinuously. On the strand that has anti-fork orientation (the lagging strand), the newDNA is synthesizeddiscontinuouslyas a series of shortOkazaki fragmentsthat are then joint together. 17

  18. Semi-discontinuously replication Continously synthesis 18 Discontinously synthesis

  19. Bi-directionally Replication Replicon is a unit of the genome in which DNA is replicated; contains an origin for initiation of replication. Replication bubble 19

  20. Bi-directionally Replication 20

  21. The bacterial genome is a single circular replicon. 21

  22. When a replicon is circular, the presence of an eye forms the θ-structure Ori 22

  23. Each eukaryotic chromosome contains many replicons 23

  24. ori ori ori ori 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Replicon

  25. Fidelity mechanism of DNA replication (1) To obey the base-pairing rules strictly (2) DNA pol can recognize and select the correct base during the process of replication (3) DNA pol has the ability to proof-reading 25

  26. Primers are needed during the process of replication Because the DNA pol can’t catalyze two free deoxynucleotides to condense together to form the first 3’-5’ phospho-diester bond. 3’ 5’ NMP-OH 5’ dNTP 26

  27. 3 3´ 5´ 3´ 3´ 5´ 5 Uncoil direction 3 5 Primer 27

  28. Section Two Eukaryotic DNA Replication

  29. Cell Cycle G1phase: gap, the time is different, some cells in vivo, such as neurons, stop dividing completely and are said to be quiescent, locked in a G0 phase. S phase: the chromosomal DNA is replicated G2 phase: cells prepare for mitosis. M phase: mitosis and cell division occur. 29

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  31. The requirements of DNA replication (1) Precursors 5’-triphosphate deoxyribonucleoside (dNTPs) (2) Template both strands of parental DNA (3)DNA polymerases (DNA pol) (4) Primer a short fragment of RNA to complementary to one strand of parental DNA (5)Accessory proteinsand enzymes 31

  32. (dNMP)n + dNTP (dNMP)n+1 + PPi 2.1 Enzymes and Proteins involved in Eukaryotic DNA Replication 2.1.1 DNA Polymerases DNA polymerases catalyze the addition of nucleotides to 3’-end of preexisting DNA strand during DNA synthesis. The reaction catalyzed by DNA polymerase is: 32

  33. 3’ P P P P P P C G template G C A A A T T A T P P P P P P OH T 5’ Mechanism of DNA replication 5’ The direction of new strand 3’ 33

  34. (dNMP)n+ dNTP → (dNMP)n+1+ PPi Template：3’ → 5’ New strand：5’ → 3’ Base pairing：A-T，G-C Primer: RNA 34

  35. P E Activities of DNA polymerases ( DNA-pol ) (DNA-dependent DNA polymerase, DDDP ) 5’ →3’ polymerization activity exonuclease activity : 5’ →3’ exonuclease activity 3’ →5’ exonuclease activity Mg2+ and Zn2+ are needed for the actions of DNA pol 35

  36. Exonuclease activity : 5´ A G C T T C A G G A T A 3´ | | | | | | | | | | | ? 3´ T C G A A G T C C T A G C G A C 5´ 3’ →5’ exonuclease activity: to excise bases added to DNA incorrectly. Proof-reading activity 5’ →3’ exonuclease activity: to excises the mutant DNA fragment Mutant repairing 36

  37. The choice of the nucleotide to add to the strand is dictated by base pairing with the template strand. 37

  38. DNA polymerase in eukaryotes There are several DNA polymerases in eukaryotes, , , , , , et al. 38

  39. DNA polymerasescarries aprimase subunit and has the activity of primase, so it can make primer. DNA polymerasessynthesizes the leading strand and lagging strand, as well as has3′→5′exonuclease activity and so can proof-read the DNA made. 39

  40. DNA polymerases  and  replicate chromosomal DNA.  DNA polymerases  and  repair DNA. DNA polymerases  replicates mitochondrial DNA. 40

  41. OH 2.1.2 Primase Primase can make RNA (8~10 nt) directly on the single-stranded DNA template because, like all RNA polymerase, it does not require a primer to begin synthesis. 41

  42. 2.1.3 DNA helicase Role:to unwind the double helix (using ATP as energy source). The DNA template is a double helix with each strand wound tightly around each other and hence thetwo strands must be unwound during replication. Helicases are a class of enzymes vital to all living organisms. They are motor proteins that move directionally along a nucleic acidphosphodiester backbone, separating two annealed nucleic acid strands (i.e. DNA, RNA, or RNA-DNA hybrid) using energy derived from ATPhydrolysis. 42

  43. 2.1.4 Single-stranded DNA-binding protein (SSB ) Role: To prevent the single-stranded regions from base-pairing again. Single-strand binding protein, also known as SSB or SSBP, binds single stranded regions of DNA to prevent premature reannealing. The strands have a natural tendency to revert to the duplex form, but SSB binds to the single strands, keeping them separate and allowing the DNA replication machinery to perform its function. 43

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  45. 2.1.5 DNA topoisomerase Role: To prevent the DNA double helix from supercoiled and twist. Topoisomerase I Topoisomerase II 45

  46. Topoisomerase I Cut only one strand of DNA, no ATP needed Topoisomerase I It breaks a phosphodiester bond in one DNA strand (a single-strand break) a small distance ahead of the fork, allowing the DNA to rotate freely(swivel) around the other(intact) strand. The phosphodiester bond is then re-formed by the topoisomerase. 46

  47. Topoisomerase II Cut two strands of DNA, ATP needed Topoisomerase II It breaks a phosphodiester bond in each strand (a double-strand break) of a double-strand DNA molecule. Thus topoisomerase II binds to one double-strand DNA circle and causes a transient double-strand break that acts as a ’gate’ through which the other DNA circle can pass. Topoisomerase II then re-seals the strand breaks. 47

  48. 2.1.6 Proliferating Cell Nuclear Antigen and Replication Factor C PCNA was originally identified as an antigen that is expressed in the nuclei of cells during the DNA synthesis phase. PCNA helps hold DNA Pol δ to DNA. PCNA is clampedto DNA through the action of replication factor C (RFC). Expression of PCNA is under the control of E2Ftranscription factor-containing complexes. 48

  49. Structure of sliding clamp in mammalian cells, a trimer of PCNA, DNA passes freely through a large hole in center of the complex 49

  50. 2.1.7 RNase H and Flap Endonuclease I Flap Endonuclease I (FEN I, 侧翼内切核酸酶） Functions To remove primer from 5’-end of DNA strand RNase H degrades the RNA primer, leaving a single ribonucleotide attached to the end of a Okazaki fragment. FEN I is required to complete the removal of the last ribonucleotide. 50

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