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DNA REPLICATION

DNA REPLICATION. YILDIRIM BEYAZIT UNIVERSITY FACULTY OF MEDICINE THE DEPARTMENT OF MEDICAL BIOLOGY ASST. PROF. DR. ENDER ŞİMŞEK. DNA Replication. Process of duplication of the entire genome prior to cell division. DNA Replication.

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DNA REPLICATION

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  1. DNA REPLICATION YILDIRIM BEYAZIT UNIVERSITY FACULTY OF MEDICINE THE DEPARTMENT OF MEDICAL BIOLOGY ASST. PROF. DR. ENDER ŞİMŞEK

  2. DNA Replication Process of duplication of the entire genome prior to cell division.

  3. DNA Replication In eukaryotes , replication only occurs during the S phaseof the cell cycle and replication rate is slower resulting in a higher accuracy of replication.

  4. The mechanism of DNA replication • Three Important Steps of DNA Replication: 1. Initiation 2. Elongation 3. Termination

  5. The mechanism of DNA replication • Initiation • Proteins bind to DNA and open up double helix • Prepare DNA for complementary base pairing

  6. The mechanism of DNA replication • Initiation • Proteins bind to DNA and open up double helix • Prepare DNA for complementary base pairing • Elongation • Proteins connect the correct sequences of nucleotides into a continuous new strand of DNA

  7. The mechanism of DNA replication • Initiation • Proteins bind to DNA and open up double helix • Prepare DNA for complementary base pairing • Elongation • Proteins connect the correct sequences of nucleotides into a continuous new strand of DNA • Termination • Proteins release the replication complex

  8. Features of DNA Replication • DNA replication is semiconservative • Each strand of template DNA is being copied.

  9. Features of DNA Replication • DNA replication is semiconservative • Each strand of template DNA is being copied. • DNA replication is semidiscontinuous • The leading strand copies continuously • The lagging strand copies in segments which must be joined

  10. Features of DNA Replication • DNA replication is semiconservative • Each strand of template DNA is being copied. • DNA replication is semidiscontinuous • The leading strand copies continuously • The lagging strand copies in segments which must be joined • DNA replication is bidirectional • Bidirectional replication involves two replication forks, which move in opposite directions

  11. Flow of Genetic Information in the Cell • Mechanisms by which information is transferred in the cell is based on “Central Dogma”

  12. Proposed Models of DNA Replication • In the late 1950s, three different mechanisms were proposed for the replication of DNA;

  13. 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

  14. 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 DNA replication

  15. 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 DNA replication • Dispersive model • Parental and daughter DNA are interspersed in both strands following DNA replication

  16. Three Models for DNA Replication Semiconservative • Both parental strands stay together after DNA replication Conservative • The double-stranded DNA contains one parental and • one daughter strand following DNA replication Dispersive • Parental and daughter DNA are interspersed in both strands following DNA replication

  17. How Is DNA Replicated? Meselson and Stahl showed semiconservative replication was the correct model for the replication of DNA. They used density labeling to distinguish parent DNA strands from new DNA strands. DNA was labeled with 15N, making DNA more dense.

  18. Matthew Meselson and Franklin Stahl experiment in 1958 • Grow E. coli in the presence of 15N (a heavy isotope of Nitrogen) for many generations (The cells get heavy-labeled DNA).

  19. Matthew Meselson and Franklin Stahl experiment in 1958 • Grow E. coli in the presence of 15N (a heavy isotope of Nitrogen) for many generations (The cells get heavy-labeled DNA). • Switch to medium containing only 14N (a light isotope of Nitrogen).

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

  21. Matthew Meselson and Franklin Stahl experiment in 1958 • Grow E. coli in the presence of 15N (a heavy isotope of Nitrogen) for many generations (The 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 CsClgradient.

  22. The Meselson- Stahl Experiment (Part 1)

  23. The Meselson- Stahl Experiment (Part 2) 2ND GENERATION 1ST GENERATION

  24. DNA 14N 15N CsCl Density Gradient Centrifugation

  25. 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

  26. How Is DNA Replicated? Results of their experiment explained by the semiconservative model: If conservative, the first generation of individuals would have been both high and low density, but not intermediate.

  27. How Is DNA Replicated? Results of their experiment explained by the semiconservative model: If conservative, the first generation of individuals would have been both high and low density, but not intermediate. If dispersive, density in the first generation would be half, but this density would not appear in subsequent generations.

  28. 1955: Arthur Kornberg • Worked with E. coli. • Discovered the mechanisms of DNA synthesis. • Components are required: • dNTPs: dATP, dTTP, dGTP, dCTP (deoxyribonucleoside 5’-triphosphates) (sugar-base + 3 phosphates) 2. DNA template • DNA polymerase I (Kornberg enzyme) • Mg 2+ (optimizes DNA polymerase activity) • PRIMERS!!!

  29. How Is DNA Replicated? Two steps in DNA replication: The double helix is unwound, making two template strands.

  30. How Is DNA Replicated? Two steps in DNA replication: The double helix is unwound, making two template strands. New nucleotides are added to the new strand at the 3′ end and joined by phospho-diesterlinkages. (Sequence is determined by complementary base pairing.)

  31. Each New DNA Strand Grows from Its 5´ End to Its 3´ End New nucleotides are added to the new strand at the 3′ end and joined by phospho-diester linkages.

  32. Each New DNA Strand Grows from Its 5´ End to Its 3´ End DNA chain elongation catalyzed by DNA polymerase

  33. How Is DNA Replicated? All chromosomes have a region called origin of replication (ori). Proteins in the replication complex bind to a DNA sequence in ori.

  34. How Is DNA Replicated? *** DNA replication begins with a short primer— a starter strand. *** The primer is complementary to the DNA template. *** Primase—an enzyme—synthesizes DNA one nucleotide at a time. *** DNA polymerase adds nucleotides to the 3′ end.

  35. How Is DNA Replicated?

  36. DNA Polymerase Binds to the Template Strand ***DNA polymerases are larger than their substrates, the dNTPs, and the template DNA. ***The enzyme is shaped like an open right hand—the “palm” brings the active site and the substrates into contact. ***The “fingers” recognize the nucleotide bases.

  37. How Is DNA Replicated? Other proteins play a role in replication as well; DNA helicaseuses energy from ATP hydrolysis to unwind the DNA. Single-strand DNA binding proteins keep the strands from getting back together.

  38. How Is DNA Replicated? The replication fork is the site where DNA unwinds to expose bases. One new strand, the leading strand, is oriented to grow at its 3′ end as the fork opens. The lagging strand is oriented so that its exposed 3′ end gets farther from the fork.

  39. How Is DNA Replicated? Synthesis of the lagging strand occurs in small, discontinuous stretches—Okazaki fragments. Each Okazaki fragment requires its own primer, synthesized by the primase. DNA polymerase III adds nucleotides to the 3′ end, until reaching the primer of the previous fragment.

  40. How Is DNA Replicated? DNA polymerase I then replaces the primer with DNA. The final phospho-diesterlinkage between fragments is catalyzed by DNA ligase.

  41. How Is DNA Replicated? Small, circular chromosomes have a single origin of replication. As DNA moves through the replication complex, two interlocking circular chromosomes are formed. DNA topoisomerase separates the two chromosomes.

  42. How Is DNA Replicated? Newly replicated strand is stabilized by a sliding DNA clamp (a protein). The sliding DNA clamp was recognized in dividing cellscalled the proliferating cell nuclear antigen (PCNA).

  43. A Sliding DNA Clamp Increases the Efficiency of DNA Polymerization!!!

  44. Important Enzymes in DNA Replication

  45. Eukaryotic Enzymes: Five common DNA polymerases from mammals. Polymerase  (alpha): nuclear, DNA replication, no proofreading Polymerase  (beta): nuclear, DNA repair, no proofreading Polymerase  (gamma): mitochondria, DNA replication, proofreading Polymerase  (delta): nuclear, DNA replication, proofreading Polymerase  (epsilon): nuclear, DNA repair (?), proofreading

  46. How Is DNA Replicated? Large linear chromosomes have many origins of replication. Replication complexes bind to the sites at the same time and catalyze simultaneous replication.

  47. Origin of replication (e.g., the prokaryote example): • Begins with double-helix denaturing into single-strands thus exposing the bases. • Exposes a replication bubble from which replication proceeds in both directions. ~245 bp in E. coli

  48. Fig. 3.9 Bidirectional replication of circular DNA molecules

  49. Figure 13.19 Replication of Small Circular and Large Linear Chromosomes (B)

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