Nucleotide excision repair

1
Nucleotide excision repair
Repairs UV-induced damage e.g. thymidine
dimers Complex process in eukaryotes, over 30
enzymes involved. Number of disease processes
e.g. Cockaynes syndrome and XPD Much simpler in
prokaryotes 4 enzymes Uvr A-D Uvr A 106kDa
ATPase Uvr B 76kDa Helicase homology cryptic
ATPase Uvr C 69.4kDa Uvr D 82kDa DNA
Helicase
2
Repair sequence
A
A

• Uvr A forms dimers in solution
• Uvr B binds to Uvr A2
• Uvr A2B searches for lesions
• When a lesion is found Uvr B docks onto the
  lesion site
• UvrA2 is kicked off the DNA
• Uvr C binds cutting a 12bp fragment around the
  lesion site

3
Repair sequence

• Uvr A forms dimers in solution
• Uvr B binds to Uvr A2
• Uvr A2B searches for lesions
• When a lesion is found Uvr B docks onto the
  lesion site
• UvrA2 is kicked off the DNA
• Uvr C binds cutting a 12bp fragment around the
  lesion site
• Uvr D excises the 12bp oligo and Uvr B
• DNA pol fills in the gap and ligase seals the
  nicks

4
Strategy

• Label UvrB with an HA (haemagluttinin) tag
• Bind through antibodies to a Qdot

UvrB
HA
Y
Y
570
655
5
Using Q-dot 655 conjugate
UvrA -


HA-WTUvrB - -
HA-UvrB?4


HA antibody
-
-
Q-dot secondary
- - 12 11 1.51 21
- - 12 11 1.51 21 51
UvrB-HA antibody-DNA
UvrA-DNA
UvrB-DNA
Free DNA
UvrA 20 nM, UvrB 100 nM, HA antibody 100
nM DNA 1 nM 5 F50/NDB50, the ratio in the
figure is molar ratio of Q-dot secondary HA
primary antibody, 1 agarose, 100 V for 1 hr in
cold room
Q-dot-antibody-UvrB-DNA complexes
6
Single molecule approach
7
Actual Photos

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