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Outline for Replication section of BIO 319 Fall 2007

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  1. Outline for Replicationsection of BIO 319 Fall 2007 Chapter 11 Problems: C8, C16, C24, C28, C30 I. General Features of Replication A. Semi-Conservative B. Starts at Origin C. Bidirectional D. Semi-Discontinuous II. Identifying Proteins and Enzymes of Replication III. Detailed Examination of the Mechanism of Replication A. Initiation B. Priming C. Elongation D. Proofreading and Termination

  2. 5’ 3’ Identical base sequences 5’ 3’ 3’ 5’ 5’ 3’ Watson/Crick proposed mechanism of DNA replication Figure 11.1

  3. Proposed Models of DNA Replication • In the late 1950s, three different mechanisms were proposed for the replication of DNA • Conservative model • Both parental strands stay together after DNA replication • Semiconservative model • The double-stranded DNA contains one parental and one daughter strand following replication • Dispersive model • Parental and daughter DNA are interspersed in both strands following replication

  4. Figure 11.2 Three models for DNA replication

  5. Matthew Meselson and Franklin Stahl experiment in 1958 • Grow E. coli in the presence of 15N (a heavy isotope of Nitrogen) for many generations • Cells get heavy-labeled DNA • Switch to medium containing only 14N (a light isotope of Nitrogen) • Collect sample of cells after various times • Analyze the density of the DNA by centrifugation using a CsCl gradient

  6. DNA 14N 15N CsCl Density Gradient Centrifugation

  7. Generation 15N 0 Shift cells to 14N 1 2 3

  8. Interpreting the Data After one generation, DNA is “half-heavy” After ~ two generations, DNA is of two types: “light” and “half-heavy” This is consistent with only the semi-conservative model

  9. BACTERIAL REPLICATION • DNA synthesis begins at a site termed the origin of replication • Each bacterial chromosome has only one • Synthesis of DNA proceeds bidirectionally around the bacterial chromosome • eventually meeting at the opposite side of the bacterial chromosome • Where replication ends

  10. Figure 11.4 Overview of bacterial DNA replication

  11. Autoradiography: Radioactivity darkens film Radioactive bacterial colonies on an agar petri dish

  12. Replication Starts at an Origin and is Bidirectional

  13. Directionality of the DNA strands at a replication fork Fork movement Lagging strand Leading strand

  14. Experimental demonstration of semi-discontinuous Replication 3H labeled Okazaki fragments seen in sucrose density gradients Wild-type cells DNA Ligase deficient cells Sucrose gradient Top= Smaller Bottom= Bigger

  15. 1. Genetic Approach: Obtain Mutants that are defective in Replication • Such mutations are Lethal! b. Conditional lethal c.Temperature sensitive (ts) lethal 2. Method: a. Mutagenize cells b. Plate the cells on agar plates and grow at 30 oC c. Replica plate and grow at 37oC II. Identifying Proteins and Enzymes involved in Replication A. Combine Genetics and Biochemistry

  16. 3. Identifying which ts lethal mutants have defects in Replication a. pick ts colonies from 30oC plate and grow them in liquid medium at 30oC. b. shift them to 37oC c. add Bromodeoxyyridine (BrdU) and continue growth for a short time at 37oC d. remove the BrdU and irradiate the cells with UV light 1). if BrdU is incorporated into the DNA the UV light will kill the cells e. return the cells to 30 oC f. the cells that revive and continue to grow did not incorporate BrdU because they have a defect in Replication!!! g. Those ts cells that had other defects continued to replicate their DNA and incorporate BrdU, and hence the UV light killed them.

  17. DNA Replication In Vitro • The in vitro study of DNA replication was pioneered by Arthur Kornberg • Nobel Prize in 1959 Kornberg mixed the following • An extract of proteins from E. coli • Template DNA • Radiolabeled nucleotides • Incubated to allow the synthesis of new DNA strands • Addition of acid will precipitate these DNA strands • Centrifugation will separate them from the radioactive nucleotides

  18. ts mutant ts mutant WT ts mutant WT DNA DNA DNA DNA 37C 37C 30C 37C In vitro Complementation of mutants in DNA Replication

  19. Initiation of Replication • The origin of replication in E. coli is termed oriC • origin of Chromosomal replication • Important DNA sequences in oriC • AT-rich region • DnaA boxes

  20. Figure 11.5 DNA sequences at the Bacterial origin of Replication

  21. Figure11.6 Initiation of Replication at oriC • DNA replication is initiated by the binding of DnaA proteins to the DnaA box sequences • causes the region to wrap around the DnaA proteins and separates the AT-rich region

  22. SSB SSB SSB SSB • Uses energy from ATP to unwind the duplex DNA Figure 11.6 continued

  23. DNA helicase separates the two DNA strands by breaking the hydrogen bonds between them • This generates positive supercoiling ahead of each replication fork • DNA gyrase travels ahead of the helicase and alleviates these supercoils • Single-strand binding proteins bind to the separated DNA strands to keep them apart • Then short (10 to 12 nucleotides) RNA primersare synthesized by DNA primase • These short RNA strands start, or prime, DNA synthesis

  24. Fig. 11.9a(TE Art) DNA Polymerase Cannot Initiate new Strands 5’ Unable to covalently link the 2 individual nucleotides together 3’ 5’ 5’ 3’ 5’ Able to covalently link together 3’ 3’ 5’ Primer 5’ 3’

  25. Figure 11.10 Innermost phosphate 11-30

  26. DNA Polymerase III- does the bulk of copying DNA in Replication

  27. Figure 11.8 Schematic representation of DNA Polymerase III Structure resembles a human right hand Template DNA thread through the palm; Thumb and fingers wrapped around the DNA

  28. Direction of synthesis on leading strand 3’ 5’ 3’ 5’ 3’ Direction of synthesis on lagging strand 5’ Figure 11.7 Two dimensional view of a replication fork

  29. Figure 11.13 “Three Dimensional” view of Replication Fork Direction of synthesis of leading strand Direction of synthesis Of lagging strand Direction of fork movement

  30. Proofreading by the 3’  5’ exonuclease activity of DNA polymerases during DNA replication.

  31. Knicks are something else all together! Nicks are single strand breaks in double stranded DNA

  32. Synthesis and replacement of RNA primers during DNA replication

  33. DNA polymerases can only synthesize DNA only in the 5’ to 3’ direction and cannot initiate DNA synthesis • These two features pose a problem at the 3’ end of linear chromosomes Figure 11.24 Problem at ends of eukaryotic linear Chromosomes

  34. If this problem is not solved • The linear chromosome becomes progressively shorter with each round of DNA replication • The cell solves this problem by adding DNA sequences to the ends of chromosome: telomeres • Small repeated sequences (100-1000’s) • Catalyzed by the enzyme telomerase • Telomerase contains proteinand RNA • The RNA functions as the template • complementary to the DNA sequence found in the telomeric repeat • This allows the telomerase to bind to the 3’ overhang

  35. The complementary strand is made by primase, DNA polymerase and ligase Step 1 = Binding The binding-polymerization-translocation cycle can occurs many times Step 2 = Polymerization This greatly lengthens one of the strands Step 3 = Translocation Figure 11.25 RNA primer 11-80

  36. Viral Lifecycle of a Retrovirus (HIV)