DNA repair

1
Topoisomerase relieves supercoilingnicks and
reaneals one strand of DNA.
DNA B a helicase that separates ds DNA into ss
DNA via ATP hydrolysis
Figure 30-28 The replication of E. coli DNA.
2
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
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).
Page 1141
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

Source Lehninger pg. 940
8
Figure 30-20 The reactions catalyzed by E. coli
DNA ligase.
Page 1150
9
Figure 30-21 X-Ray structure of DNA ligase from
Thermus filiformis.
Page 1151
10
Quick Comparison of DNA polymerases I and III Quick Comparison of DNA polymerases I and III 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 ß 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.
11
(No Transcript)
12
DNA Pol III holoenzyme.
13
Figure 30-13b The ? subunit of E. coli Pol III
holoenzyme. Space-filling model of sliding clamp
in hypothetical complex with B-DNA.
Page 1146
14
Sliding clamp
http//www.callutheran.edu/Academic_Programs/Depar
tments/BioDev/omm/poliiib_2/poliiib.htm
15
Heres a computer modelof DNA replicationhttp/
/www.youtube.com/watch?v4jtmOZaIvS0
This is a pretty good outline http//www.youtube.
com/watch?vteV62zrm2P0NR1
Another one with review questions (perhaps
oversimplified) http//www.wiley.com/college/pratt
/0471393878/student/animations/dna_replication/ind
ex.html
16
FIDELITY OF REPLICATION

• Expect 1/103-4, get 1/108-10.
• 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

• http//www.nature.com/nature/journal/v421/n6921/fu
  ll/nature01408.html

18
DNA repair

• Ilkka Koskela
• Katri Vilkman

19
Foreword

• DNA
• variation is an essential factor to evolution
  (1000-106 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 -gt 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.
• -gtensures that DNA damages are repaired before
  the cell divides

29
References

• Pictures
• http//www.2modern.com/index.asp?PageActionVIEWPR
  ODProdID985
• http//www.senescence.info/WS.jpg
• http//en.wikipedia.org/wiki/Dna
• http//www.funpecrp.com.br/gmr/year2003/vol1-2/ima
  gens/sim0001fig1.jpg
• http//www.science.siu.edu/microbiology/micr460/Pa
  geMill20Images/image32.gif
• http//www.bio.brandeis.edu/haberlab/jehsite/image
  s/nhejd.gif
• http//www.biochemsoctrans.org/bst/029/0655/bst029
  0655f02.gif
• http//www.antigenics.com/products/tech/hsp/images
  /animation.jpg
• http//bioinformatics.psb.ugent.be/images/illust_c
  ell_cycle_large.jpg