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DNA Metabolism

DNA Metabolism. DNA replication: processes by which copies of DNA molecules are faithfully made. DNA repair: processes by which the integrity of DNA are maintained. DNA recombination: processes by which the DNA sequences are rearranged. Map of the E. coli chromosome.

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DNA Metabolism

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  1. DNA Metabolism • DNA replication: processes by which copies of DNA molecules are faithfully made. • DNA repair: processes by which the integrity of DNA are maintained. • DNA recombination: processes by which the DNA sequences are rearranged.

  2. Map of the E. coli chromosome.

  3. DNA Replication Is Semiconservative.

  4. Replication Forks may Move Either Unidirectionally or Bidirectionally

  5. Replication Begins at an Origin and Proceeds Bidirectionally in Many Bacteria Such as E. coli.

  6. DNA synthesis is catalyzed by DNA polymerases in the presence of (i) primer, (ii) template, (iii) all 4 dNTP, and (iv) a divalent cathion such as Mg++.

  7. DNA Synthesis Can’t be Continuously on Both Strands (because the DNA duplex is antiparallel and all DNA polymerases synthesize DNA in a 5’ to 3’ direction) What is the source of primer used for lagging strand synthesis?

  8. DNA Replication is Very Accurate • Base selection by DNA polymerase is fairly accurate (about 1 error per 104) • Proofreading by the 3’ to 5’ exonuclease associated with DNA polymerase improves the accuracy about 102 to 103-fold. • Mismatch repair system repairs any mismatched base pairs remaining after replication and further improves the accuracy.

  9. An Example of Proofreading by the 3’ to 5’ Exonuclease of DNA Polymerase I of E. coli

  10. Large (Klenow) fragment of DNA polymerase I retains polymerization and proofreading (3’ to 5’ exo)

  11. DNA polymerase I has 5’ to 3’ exonuclease and can conduct Nick Translation

  12. Holoenzyme consists of two cores, two b subunits and one g complex held together by a dimer of t. So it is an asymmetric dimer.

  13. DNA polymerase III

  14. The two b subunits of PolIII form a circular clamp that surrounds DNA

  15. DNA Replication requires many enzymes and protein factors • Helicases: separation of DNA duplex. • Topoisomerase: relieves topological stress • Single-strand DNA binding proteins: stabilizes separated DNA strands. • Primase: synthesizes RNA primer. • DNA Pol I: removes RNA in Okazaki fragments and fills the gaps between Okazaki fragments. • Ligase: seals nicks.

  16. Replication of the E. coli chromosome • Initiation. • Elongation. • Termination.

  17. Initiation begins at a fixed origin, called oriC, which consists of 245 bp bearing DNA sequences that are highly conserved among bacterial replication origins.

  18. Model for initiation of replication at oriC.

  19. Proteins involved in Elongation of DNA

  20. Elongation:Synthesis of Okazaki fragments

  21. Model for the synthesis of DNA on the leading and lagging strands by the asymmetric dimer of PolIII

  22. Pol I can remove RNA primer and synthesize DNA to fill the gap

  23. Termination: When the two opposing forks meet in a circular chromosome. Replication of the DNA separating the opposing forks generated catenanes, or interlinked circles.

  24. Termination sequences and Tus (termination utilization substance) can arrest a replication fork

  25. Replication in eukaryotic cells is more complex • Contains many replicons. • How is DNA replication initiated in each replicon is not well understood. Yeast cells appears to employ ARS (autonomously replicating sequences) and ORC (origin recognition complex) to initiate replication. • More than one DNA polymerase are used to replicate DNA. • End-replication problem of linear DNA.

  26. DNA Repair • DNA damage may arise: (i) spontaneously, (ii) environmental exposure to mutagens, or (iii) cellular metabolism. • DNA damage may be classified as: (I) strand breaks, (ii) base loss (AP site), (iii) base damages, (iv) adducts, (v) cross-links, (vi) sugar damages, (vii) DNA-protein cross links. • DNA damage, if not repaired, may affect replication and transcription, leading to mutation or cell death.

  27. Ames test for mutagens (carcinogens)

  28. Methylataion and Mismatch Repair

  29. Model for Mismatch Repair

  30. Base-Excision Repair

  31. Nucleotide-Excision Repair in E. coli and Humans

  32. Direct Repair: Photoreactivation by photolyase

  33. Alkylation of DNA by alkylating agents

  34. O6-methyl G, if not repaired, may produce a mutation

  35. Direct Repair:Reversal of O6 methyl G to G by methyltransferase

  36. Direct repair of alkylated bases by AlkB. Direct re

  37. Effect of DNA damage on replication: (i) coding lesions won’t interfere with replication but may produce mutation, (ii) non-coding lesions will interfere with replication and may lead to formation of daughter-strand gaps (DSG) or double-strand breaks (DSB). DSG and DSB may be repaired by recombination process, to be discussed in the following section.

  38. DNA repair and cancer • Defects in the genes encoding the proteins involved in nucleotide-excision repair, mismatch repair, and recombination repair have all been linked to human cancer. • Examples are: (i) xeroderma pigmentosum (or XP) patients with defects in nucleotide-excision repair, (ii) HNPCC (hereditary nonpoplyposis colon cancer) patients with defects in hMLH1 and hMSH2, and (3) breast cancer patients with inherited defects in BRCA1 and Brca2, which are known to interact with Rad 51 (the eukaryotic homolog of RecA) and therefore may have defective recombination repair.

  39. DNA Recombination • Homologous recombination or generalized recombination. • Site-specific recombinataion. • Transposition.

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