1. Figure 30-28 The replication of E. coli DNA.
Topoisomerase: relieves supercoiling—nicks and reaneals one strand of DNA.
DNA B: a helicase that separates ds DNA into ss DNA via ATP hydrolysis
Figure The replication of E. coli DNA.

2. Produced from subtilisin or trypsin cleavage
Functional domains in the Klenow Fragment (left) and DNA Polymerase I (PDB).
Produced from subtilisin or trypsin cleavage
Retains polymerase and 3’-5’ exo activity

3. Page 1141
Figure 30-8b X-Ray structure of E. coli DNA polymerase I Klenow fragment (KF) in complex with a dsDNA (a tube-and-arrow representation of the complex in the same orientation as Part a).

4. The structure of the Klenow fragment of DNAP I from E. coli Fingers
Palm

5. Mismatch repair during DNA replicaiton

6. Replacing RNA primers

7. Nick Translation Requires 5’-3’ activity of DNA pol I Steps
At a nick (free 3’ OH) in the DNA the DNA pol I binds and digests nucleotides in a 5’-3’ direction
The DNA polymerase activity synthesizes a new DNA strand
A nick remains as the DNA pol I dissociates from the ds DNA.
The nick is closed via DNA ligase
Requires 5’-3’ activity of DNA pol I
Steps
At a nick (free 3’ OH) in the DNA the DNA pol I binds and digests nucleotides in a 5’-3’ direction
The DNA polymerase activity synthesizes a new DNA strand
A nick remains as the DNA pol I dissociates from the ds DNA.
The nick is closed via DNA ligase
Uses:
removal of RNA primers
DNA repair
Great for DNA labeling with radioactive dNTPs
Source: Lehninger pg. 940

8. Figure 30-20 The reactions catalyzed by E. coli DNA ligase.
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9. Figure 30-21 X-Ray structure of DNA ligase from Thermus filiformis.
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10. Quick Comparison of DNA polymerases I and III
DNA polymerase III
DNA polymerase I
 Structure
asymmetric dimer; i. e., two cores with other accessory subunits. It can thus move with the fork and make both leading and lagging strands.
monomeric protein, 3 active sites. 5'-to-3' exonuclease and polymerase on the same molecule for removing RNA primers is effective and efficient.
 Activities
Polymerization and 3'-to-5' exonuclease, but on different subunits. This is the replicative polymerase in the cell. Can only isolate conditional-lethal dnaE mutants. Synthesizes both leading and lagging strands. No 5' to 3' exonuclease activity.
Polymerization, 3'-to-5' exonuclease, and 5'-to-3' exonuclease (mutants lacking this essential activity are not viable). Primary function is to remove RNA primers on the lagging strand, and fill-in the resulting gaps.
 Vmax (nuc./sec)
250-1,000 nucleotides/second. Only this polymerase is fast enough to be the main replicative enzyme.
20 nucleotides/second. Capable of "filling in" DNA to replace the short (about 10 nucleotides) RNA primers on Okazaki fragments.
 Processivity
Highly processive. The β subunit is a sliding clamp. The holoenzyme remains associated with the fork until replication terminates.
Pol I does NOT remain associated with the lagging strand, but disassociates after each RNA primer is removed.
 Molecules/cell
10-20 molecules/cell. In rapidly growing cells, there are 6 forks. If one processive holoenzyme (two cores) is at each fork, then only 12 core polymerases are needed for replication.
About 400 molecules/cell. Higher concentration means that it can reassociate with the lagging strand easily.

12. DNA Pol III holoenzyme.

13. Figure 30-13b. The  subunit of E. coli Pol III holoenzyme
Figure 30-13b The  subunit of E. coli Pol III holoenzyme. Space-filling model of sliding clamp in hypothetical complex with B-DNA.
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14. Sliding clamp

15. Here’s a computer model of DNA replication http://www. youtube
This is a pretty good outline:
Another one with review questions (perhaps oversimplified)

16. FIDELITY OF REPLICATION
Expect 1/103-4, get 1/
Factors
3’5’ exonuclease activity in DNA pols
Use of “tagged” primers to initiate synthesis
Battery of repair enzymes
Cells maintain balanced levels of dNTPs
Expect 1/103-4, get 1/
Factors
3’5’ exonuclease activity in DNA pols
Use of “tagged” primers to initiate synthesis
Battery of repair enzymes
Cells maintain balanced levels of dNTPs

17. This article is a simple overview of repair processes

18. Ilkka Koskela Katri Vilkman
DNA repair
Ilkka Koskela
Katri Vilkman

19. Foreword
DNA
variation is an essential factor to evolution ( ^6 lesions per day)
stability is important for the individual (less than 1/1000 mutations are permanent)
A relatively large amount of genes are devoted to coding DNA repair functions.

20. Sources of damage:
heat
metabolic accidents (free radicals)
radiation (UV, X-Ray)
exposure to substances (especially aromatic compounds)
Types of damage:
deamination of nucleotides
depurination of nucleotides
oxidation of bases
breaks in DNA strands

21. Diseases colon cancer cellular ultraviolet sensitivity
Werner syndrome (premature aging, retarded growth)
Bloom syndrome (sunlight hypersensitivity)

22. Damage of the double helix
Single strand damage
information is still backed up in the other strand
Double strand damage
no backup
can cause the chromosome to break up

23. Single strand repair Base excision repair
A base-specific DNA glycosylase detects an altered base and removes it
AP endonuclease and phosphodiesterase remove sugar phosphate
DNA Polymerase fills and DNA ligase seals the nick

24. Single strand repair Nucleotide excision repair
a large multienzyme compound scans the DNA strand for anomalities
upon detection a nuclease cuts the strand on both sides of the damage
DNA helicase removes the oligonucleotide
the gap is repaired by DNA polymerase and DNA ligase enzymes

25. Double strand repair Nonhomologous end-joining
only in emergency situations
two broken ends of DNA are joined together
a couple of nucleotides are cut from both of the strands
ligase joins the strands together

26. Double strand repair Homologous end-joining
damaged site is copied from the other chromosome by special recombination proteins

27. DNA repair enzymes
a lot of DNA damage -> elevated levels of repair enzymes
extreme change in cell's environment (heat, UV, radiation) activates genes that code DNA repair enzymes
For an example, heat-shock proteins are produced in heat-shock response when being subjected to high temperatures.

28. Cell Cycle and DNA repair
Cell cycle is delayed if there is a lot of DNA damage.
Repairing DNA as well as signals sent by damaged DNA delays progression of cell cycle.
->ensures that DNA damages are repaired before the cell divides

29. References
Pictures