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DNA Metabolism

DNA Metabolism. DNA replication: processes which DNA is being faithfully duplicated. DNA recombination: processes which the nucleotide sequence of DNA is being rearranged. DNA repair: processes which the structural integrity of DNA is being maintained. Objectives of DNA replication.

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DNA Metabolism

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  1. DNA Metabolism • DNA replication: processes which DNA is being faithfully duplicated. • DNA recombination: processes which the nucleotide sequence of DNA is being rearranged. • DNA repair: processes which the structural integrity of DNA is being maintained.

  2. Objectives of DNA replication • Basic mechanisms: (i) semiconservative, conservative, or random dispersive, (ii) continuous,semidiscontinuous or discontinuous, (iii) unidirectional, bidirectional, or rolling circle. • Enzymology: (i) identification of genes involved in replication , (ii) biochemical function of the protein products of these genes. • Replicon: the unit of DNA replication. A single DNA molecule may consist of one replicon (eg., in prokaryotes) or many replicons (eg., in eukaryotes). • Replication of any replicon may be separated into threephases: initiation, elongation and termination. The methods for identification of replication origin and the enzymes involved in each phase will be discussed. • Regulation of DNA replication – how cells ensure each DNA is replicated only once per cell cycle.

  3. Models of DNA replication

  4. Prediction of experimental outcomes

  5. DNA replication is semiconservative

  6. DNA synthesis is catalyzed by DNA polymerases in the presence of (i) primer, (ii) template, (iii) all 4 dNTP, and (iv) a divalent cation such as Mg++, (v) synthesis is from 5’ to 3’.

  7. Models of DNA chain elongation at replicating fork Fig. 20.5

  8. 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)

  9. Semidiscontinuous or discontinuous? Fig. 20.6 BioEssays 27:633-636 (2005)

  10. What is the source of primer used for lagging strand synthesis? Fig. 20.7

  11. RNA primers 10-12 nt long are used to synthesize Okazaki fragments Fig. 20.8

  12. Modes of DNA replication • Bubbles (eyes) and Y structures. • Theta mode (circular DNA). • Displacement loop (D-loop). • Rolling circle (Lariat or Sigma form)

  13. Bubble or eyes. Fig. 20.10

  14. Theta mode. Fig. 20.9

  15. Rolling circle replication Fig. 20.13

  16. Fig. 20.14

  17. Displacement loop Fig. 15.16

  18. Fig. 15.5

  19. Directionalityof DNA chain elongation Fig. 20.11

  20. Unidirectional replication of colicin E1 DNA Fig. 20.12

  21. Enzymology of DNA replication • Identification of genes involved in replication: (i) isolation of conditional lethal mutants (eg., temperature-sensitive mutations) that affect DNA synthesis, (ii) map and clone the gene of interest. • Biochemical function of the protein products of these genes.

  22. Fig. 20.20

  23. Fig. 20.22

  24. Proteins involved in the initiation of replication

  25. Proteins involved in the elongation of DNA

  26. Initiation of Replication • Start of DNA chains: (i) RNA primer, (ii) terminal protein primer, (iii) parental strand primer. • Identification of origins: (i) physical mapping: EM, two-D gel electrophoresis etc., (ii) genetic mapping, (iii) functional mapping by DNA cloning. • Chromosomal origins: (i) E. coli and other bacterial origins, (ii) origins without an initiator protein (ColE1 and T7), (iii) origins cleaved by initiator endonucleases, (iv) yeast autonomously replicating sequences (ARS). • Initiation from the E. coli oriC.

  27. Terminal protein may be used as primer to initiate replication Fig. 16.2

  28. Fig. 16.3

  29. Fig. 16.4

  30. Parental DNA strand as primer: Nicking by specific endonuclease to produce 3’-OH Fig. 16.5

  31. Fig. 16.7

  32. Fig. 16.11

  33. Fig. 16.12

  34. Fig. 16.8

  35. Two-dimensional gel electrophoresis to identify origin Fig. 21.5

  36. Fig. 21.6

  37. Mapping of SV40 origin by EM

  38. Fig. 21.3

  39. Functional cloning of replication origin

  40. Bacterial replication origins

  41. The yeast origins of replication are contained within autonomous replicating sequences (ARSs) that are composed of 4 regions (A, B1, B2, and B3). An 11-bp (5-[T/A]TTTAPyPuTTT[T/A]-3’) consensus sequence is highly conserved in ARSs. Fig. 21.7

  42. Fig. 21.1

  43. Elongation at a replication fork • Replication speed. • Enzymes involved in DNA elongation at a replication fork and their functions. • Model of simultaneous synthesis of both DNA strands by PolIII.

  44. Fig. 21.8

  45. Fig. 21.9

  46. Proteins involved in the elongation of DNA

  47. Elongation

  48. DNA Polymerases Processivity of DNA polymerase is determined at low enzyme concentration and in the presence of excessive substrates.

  49. PolIII*consists of two cores, a clamp-loading complex (g complex) consisting of t2gdd’, and two additional proteins c and y. Holoenzyme is PolIII* plus b subunits.

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