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Genetic Information Transfer

Genetic Information Transfer. Central dogma. replication. transcription. translation. DNA. protein. RNA. reverse transcription. Replication: synthesis of daughter DNA from parental DNA Transcription: synthesis of RNA using DNA as the template

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Genetic Information Transfer

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  1. Genetic Information Transfer

  2. Central dogma replication transcription translation DNA protein RNA reverse transcription

  3. Replication: synthesis of daughter DNA from parental DNA • Transcription: synthesis of RNA using DNA as the template • Translation: protein synthesis using mRNA molecules as the template • Reverse transcription: synthesis of DNA using RNA as the template

  4. Lecture 2 DNA Replication （DNA Biosynthesis）

  5. Section 1General Concepts of DNA Replication

  6. Double helix structure of DNA “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Watson & Crick

  7. Characteristics of replication • Semi-conservative replication • Bidirectional replication • Semi-continuous replication

  8. §1.1 Semi-Conservative Replication ——Meselson and Stahl (1958)

  9. Semiconservative replication Definition:Half of the parental DNA molecule is conserved in each new double helix, paired with a newly synthesized complementary strand. Significance:The genetic information is ensured to be transferred from one generation to the next generation with a high fidelity. A T T G C T A A C G Parent molecule A T T G C T A A C G A T T G C T A A C G Daughter molecule

  10. Examination of T7 DNA replication using electron microscopy §1.2 Bidirectional Replication Replication starts from unwinding the dsDNA at a particular point(calledorigin), followed by the synthesis on each strand. The parental dsDNA and two newly formed dsDNA form a Y-shape structure called replication fork. Origin

  11. Once the dsDNA is opened at the origin, two replication forks are formed spontaneously. These two replication forks move in opposite directions as the synthesis continue. Bidirectional replication

  12. Replication of prokaryotes • The replication process starts from the origin, and proceeds in two opposite directions. It is named  replication.

  13. Replication of eukaryotes Chromosomes of eukaryotes have multiple origins.

  14. §1.3 Semi-continuous Replication ? The DNA strands are antiparallel. At a replication fork, both strands of parental DNA serve as templates for the synthesis of new DNA; All known DNA polymerases synthesize DNA in the 5’ →3’direction but not in 3’ →5’ direction.

  15. Reiji Okazaki and his wife Tsuneko Okazaki • This dilemma was resolved by Reiji Okazaki ( in the 1960s), who found that a significant proportion of newly synthesized DNA exists as small fragments; • These units of about a thousand nucleotides are called Okazaki fragments; • They are 1000 – 2000nt long for prokaryotes and 100-150nt long for eukaryotes. 解链方向

  16. Semi-continuous replication • The leading strand :the strand synthesized continuously; • The lagging strand :the strand formed from Okazaki fragments; • Thesemi-continuous replication: Continuous synthesis of the leading strand and discontinuous synthesis of the lagging strand represent a unique feature of DNA replication. It is referred to as the semi-continuous replication.

  17. Section 2Enzymology of DNA Replication Large team of enzymes coordinates replication Let’s meetthe team…

  18. DNA replication system Template: double stranded DNA Substrate:dNTP Primer: short RNA fragment with a free 3´-OH end Enzyme:DNA-dependent DNA polymerase (DDDP), other enzymes Protein factor

  19. Daughter strand synthesis • Chemical formulation: • The nature of DNA replication is a series of 3´,5´phosphodiester bond formation catalyzed by a group of enzymes.

  20. Phosphodiester bond formation

  21. energy We comewith our ownenergy! Where’s theENERGYfor the bonding! (dNMP)n+ dNTP → (dNMP)n+1+ PPi

  22. Enzymes and protein factors

  23. §2.1 DNA Polymerase DNA-pol of prokaryotes • The first DNA- dependent DNA polymerase (short for DNA-pol I) was discovered in 1958 by Arthur Kornberg who received Nobel Prize in physiology or medicine in 1959.

  24. Kornberg liked to refer to his scientific career as a "love affair with enzymes." Arthur Kornberg (left) with his son, Roger, after Roger received the 2006 Nobel Prize in Chemistry.

  25. Later, DNA-pol II and DNA-pol III were identified in experiments using mutated E.coli cell line. • DNA-pol I possess the following biological activity. 1. 53 polymerizing 2. The 3` to 5` exonuclease activity 3. The 5` to 3` exonuclease activity • Why does a DNA polymerase also need two exonuclease activities?

  26. Proofreading and correction • DNA-pol Ihas the function to correct the mismatched nucleotides. • It identifies the mismatched nucleotide, removes it using the 3´- 5´ exonuclease activity, add a correct base, and continues the replication.

  27. Exonuclease functions 5´→3´ exonuclease activity cut primer or excise mutated segment 3´→5´ exonuclease activity excise mismatched nuleotides ？

  28. DNA-pol of E. coli

  29. Klenow fragment • Klenow fragment: large fragment (604 AA) of DNA pol I, having DNA polymerization and 3´→5´exonuclease activities, and is widely used in molecular biology.

  30. DNA-pol II • Temporary functional when DNA-pol I and DNA-pol III are not functional. • Still capable for doing synthesis on the damaged template • Participating in DNA repairing

  31. DNA-pol III • A heterodimer enzyme composed of ten different subunits • Having the highest polymerization activity (105 nt/min) • The true enzyme responsible for the elongation process

  32. DNA Polymerase III- does the bulk of copying DNA in Replication β2 subunit: sliding clamp

  33. Also called DnaG Primase (a specific RNA polymerase) : synthesize primers using free NTPs as the substrate and the ssDNA as the template. Primers: short RNA fragments (5-50 nucleotides). §2.2 Primase

  34. Dna C 解链方向 Dna B Also referred to as DnaB. It opens the double strand DNA with consuming ATP. (Zip opener) The opening process with the assistance of DnaA and DnaC §2.3 Helicase

  35. §2.4 SSB protein(single strand DNA binding protein) • maintains the DNA templatein the single strand form in order to • prevent the dsDNA formation; • protect the ssDNA degradation by nucleases.

  36. Opening the dsDNA will create supercoil ahead of replication forks, the supercoil constraint needs to be released by topoisomerases (type I and II). §2.5 Topoisomerase

  37. Topoisomerase I It cuts a phosphoester bond on one DNA strand, rotates the broken DNAfreely around the other strand to relax the constraint,and reseals the cut.

  38. Topoisomerase II It is named gyrase in prokaryotes. It cuts phosphoester bonds on both strands of dsDNA, releases the supercoil constraint, and reforms the phosphoester bonds. Antibiotics:ciprofloxacin, novobiocin and nalidixic acid, inhibit the bacterial gyrase. Anticancer agents:adriamycin, etoposide, and doxorubicin, inhibit human topoisomerase.

  39. Connect two adjacent ssDNA strands by joining the 3´-OH of one DNA strand to the 5´-P of another DNA strand. Sealing the nick in the process of replication, repairing, recombination, and splicing. §2.6 DNA Ligase 5’ 3’ 3’ HO 5’ ATP（NAD+） DNA Ligase AMP 5’ 3’ 3’ 5’

  40. Section 3DNA Replication Process

  41. Sequential actions • Initiation: recognize the starting point, separate dsDNA, primer synthesis, … • Elongation: add dNTPs to the existing strand, form phosphoester bonds, correct the mismatch bases, extending the DNA strand, … • Termination: stop the replication

  42. §3.1 Replication of prokaryotes a. Initiation • The replication starts at a particular point called origin. Genome of E. coli

  43. The structure of the origin is 248 bp long and AT-rich. DNA sequences at the Bacterial origin of Replication

  44. Formation of replication fork • DnaA recognizes origin. • DnaB(helicase) and DnaC join the DNA-DnaA complex, open the local AT-rich region, and move on the template downstream further to separate enough space. • SSB protein binds the complex to stabilize ssDNA.

  45. Primer synthesis • Primase joins and starts the synthesis of RNA primers. • Primasome: protein complex responsible for creating RNA primers on ssDNA during DNA replication. • Topoisomerase binds to the dsDNA region just before the replication forks to release the supercoil constraint.

  46. 5' 3' HO 3 primase primer 5 3 5 The short RNA fragments provide free 3´-OH groups for DNA elongation.

  47. b. Elongation • dNTPs are continuously connected to the primer or the nascent DNA chain by DNA-pol III. • The nature of the chain elongation is the series formation of the phosphodiester bonds.

  48. Lagging strand synthesis • Primers on Okazaki fragments are digested by RNase. • The gaps are filled by DNA-pol I in the 5´→3´direction. • The nick between the 5´end of one fragment and the 3´end of the next fragment issealed by DNA ligase. RNase DNA-pol I

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