1. CHAPTER 25 DNA Metabolism
Key topics:
DNA replication
DNA repair
DNA recombination

2. What is DNA Metabolism?
While functioning as a stable storage of genetic information, the structure of DNA is far from static:
A new copy of DNA is synthesized with high fidelity before each cell division
Errors that arise during or after DNA synthesis are constantly checked for, and repairs are made
Segments of DNA are rearranged either within a chromosome or between two DNA molecules giving offspring a novel DNA
DNA metabolism consists of a set of enzyme catalyzed and tightly regulated processes that achieve these tasks

3. 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.

4. Map of the E. coli chromosome.

5. DNA Replication Is Semiconservative.

6. Replication Forks may Move Either Unidirectionally or Bidirectionally

7. FIGURE 25-3b Visualization of bidirectional DNA replication
FIGURE 25-3b Visualization of bidirectional DNA replication. (b) Replication could be either unidirectional or bidirectional, and these can be distinguished by autoradiography: when tritium (3H) is added for a short period just before replication is stopped, label (pink) would be found at one or both replication forks, respectively. This technique has revealed bidirectional replication in E. coli, Bacillus subtilis, and other bacteria. The autoradiogram here shows a replication bubble from B. subtilis. The heaviest grain density (arrows) is at the two ends, where replication is occurring. The unreplicated part of the chromosome, outside the bubble, is unlabeled and thus not visible.

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

9. 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++.

10. DNA Elongation Chemistry
Parental DNA strand serves as a template
Nucleotide triphosphates serve as substrates in strand synthesis
Hydroxyl at the 3’ end of growing chain makes a bond to the -phosphorus of nucleotide
Pyrophosphate is a good leaving group

11. MECHANISM FIGURE 25-5b Elongation of a DNA chain
MECHANISM FIGURE 25-5b Elongation of a DNA chain. (b) The catalytic mechanism likely involves two Mg2+ ions, coordinated to the phosphate groups of the incoming nucleotide triphosphate and to three Asp residues, two of which are highly conserved in all DNA polymerases. The Mg2+ ion depicted on the right facilitates attack of the 3′-hydroxyl group of the primer on the α phosphate of the nucleotide triphosphate; the other Mg2+ ion facilitates displacement of the pyrophosphate. Both ions stabilize the structure of the pentacovalent transition state. RNA polymerases use a similar mechanism (see Figure 26-1b). 11

12. 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?

13. 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.

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

15. TABLE 25-1 Comparison of Three DNA Polymerases of E. coli15

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

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

18. PolIII* consists of two cores, a clamp-loading complex (g complex) consisting of t2 gdd’, and two additional proteins c and y. Holoenzyme is PolIII* plus b subunits.

19. DNA polymerase III q

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

21. 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.

22. Replication of the E. coli chromosome
Initiation.
Elongation.
Termination.

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

24. TABLE 25-3 Proteins Required to Initiate Replication at the E
TABLE 25-3 Proteins Required to Initiate Replication at the E. coli Origin

25. Model for initiation of replication at oriC.

26. Proteins involved in Elongation of DNA

27. Elongation: Synthesis of Okazaki fragments

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

29. FIGURE 25-14 (part 2) DNA synthesis on the leading and lagging strands
FIGURE (part 2) DNA synthesis on the leading and lagging strands. Events at the replication fork are coordinated by a single DNA polymerase III dimer, in an integrated complex with DnaB helicase. This figure shows the replication process already underway (parts (a) through (e) are discussed in the text). The lagging strand is looped so that DNA synthesis proceeds steadily on both the leading and lagging strand templates at the same time. Red arrows indicate the 3′ end of the two new strands and the direction of DNA synthesis. The heavy black arrows show the direction of movement of the parent DNA through the complex. An Okazaki fragment is being synthesized on the lagging strand. The subunit colors and the functions of the clamp-loading complex are explained in Figure 29

30. FIGURE 25-14 (part 3) DNA synthesis on the leading and lagging strands
FIGURE (part 3) DNA synthesis on the leading and lagging strands. Events at the replication fork are coordinated by a single DNA polymerase III dimer, in an integrated complex with DnaB helicase. This figure shows the replication process already underway (parts (a) through (e) are discussed in the text). The lagging strand is looped so that DNA synthesis proceeds steadily on both the leading and lagging strand templates at the same time. Red arrows indicate the 3′ end of the two new strands and the direction of DNA synthesis. The heavy black arrows show the direction of movement of the parent DNA through the complex. An Okazaki fragment is being synthesized on the lagging strand. The subunit colors and the functions of the clamp-loading complex are explained in Figure 30

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

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

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

35. 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.

36. Assembly of a pre-replicative complex at a eukaryotic replication origin
FIGURE Assembly of a pre-replicative complex at a eukaryotic replication origin. The initiation site (origin) is bound by ORC, CDC6, and CDT1. These proteins, many of them AAA+ ATPases, promote loading of the replicative helicase, MCM2–7, in a reaction that is analogous to the loading of the bacterial DnaB helicase by DnaC protein. Loading of the MCM helicase complex onto the DNA forms the pre-replicative complex, or pre-RC, and is the key step in the initiation of replication.

37. The End Replication Problem of Linear DNA

38. DNA Damages
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.

39. DNA Repair and Mutations
Chemical reactions and some physical processes constantly damage genomic DNA
At the molecular level, damage usually involves changes in the structure of one of the strands
Vast majority are corrected by repair systems using the other strand as a template
Some base changes escape repair and the incorrect base serves as a template in replication
The daughter DNA carries a changed sequence in both strands; the DNA has been mutated
Accumulation of mutations in eukaryotic cells is strongly correlated with cancer; most carcinogens are also mutagens

40. Ames test for mutagens (carcinogens)

41. TABLE 25-5 Types of DNA Repair Systems in E. coli

42. Methylataion and Mismatch Repair

43. Model for Mismatch Repair

45. Base-Excision Repair

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

47. Direct Repair: Photoreactivation by photolyase

48. Alkylation of DNA by alkylating agents

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

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

51. Direct repair of alkylated bases by AlkB.

52. 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.

53. TABLE 25-6 Genes Induced as Part of the SOS Response in E. coli

54. 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.
Case Files: Case 11 (Breast cancer gene) and Case 13 (Fragile X syndrome).

55. DNA Recombination Segments of DNA can rearrange their location
within a chromosome
from one chromosome to another
Such recombination is involved in many biological processes
Repair of DNA
Segregation of chromosomes during meiosis
Enhancement of generic diversity
In sexually reproducing organism, recombination and mutations are two driving forces of evolution
Recombination of co-infecting viral genomes may enhance virulence and provide resistance to antivirals

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

57. Pairing of homologous chromosomes and crossing-over in meiosis.

58. Recombination during meiosisis initiated by double-strand breaks.

59. Homologous recombination is catalyzed by enzymes
The most well characterized recombination enzymes are derived from studies with E. coli cells.
Presynapsis: helicase and/or nuclease to generate single-strand DNA with 3’-OH end (RecBCD).
Synapsis: joint molecule formation to generate Holliday juncture (RecA).
Postsynapsis: branch migration and resolution of Holliday juncture (RuvABC).

60. Helicase and nuclease activities of the RecBCD

61. RecA forms nucleoprotein filament on single-strand DNA

62. RecA filaments are extended and disassembled in the 5’ to 3’ direction
FIGURE 25-36c RecA protein. (c) After a rate-limiting nucleation step, RecA filaments are extended in the 5′→3′ direction on single-stranded DNA. Disassembly proceeds, also in the 5′→3′ direction, from the end opposite to that where extension occurs.

63. Filament assembly is assisted by RecFOR and RecX inhibits filament extension
FIGURE 25-36d RecA protein. (d) Filament assembly is assisted by the RecF, RecO, and RecR proteins (RecFOR). The RecX protein inhibits RecA filament extension. The DinI protein stabilizes RecA filaments, preventing disassembly.

64. RecA promotes joint molecule formation and strand exchange

65. Model for DNA strand exchange mediated by RecA

66. Models for recombinational DNA repair of
stalled replication fork

67. Models for recombinational DNA repair

68. Site-specific Recombination: Bacteriophage lambda integration in E
Site-specific Recombination: Bacteriophage lambda integration in E. coli

69. Effects of site-specific recombination on DNA structure

70. A site-specific recombination reaction (eg
A site-specific recombination reaction (eg. catalyzed by Int of bacteriophage lambda)

71. XerCD site-specific recombinataion system can resolve dimer into monomer

72. Immunoglobulin Genes Are Assembled by V(D)J Recombination

73. Mechanism of V(D)J Recombination

74. Transposition
Transposition is mediated by transposable elements, or transposons.
Transposons of bacteria: IS (insertion sequences) contains only sequences required for transposition and proteins (transposases) that promote the process. Complex transposons contain genes in addition to those needed for transposition.
Transposition is characterized by duplication of direct repeats (5-9 bps) at target site.
Transposition, in some instances, may be mediated through a RNA intermediate.

75. Duplication of the DNA sequence at a target site when a transposon is inserted

76. Models for Direct and Replicative Transposition

77. Replicative transposition is meidated
by a cointegrate intermediate.
Fig. 23.6

78. Fig. 23.7