Thursday, March 18, 2010

DNA Repair 3

Mechanisms of Repair: NER and BER
In the case of damaged DNA, be it via radiation, oxidation, or incorrect basepairing, it will recognize the mutation via several different mechanisms and will begin to repair the error, if it can. 

Nucleotide excision repair is one common mechanism in which the cell removes a number of nucleotides surrounding a DNA lesion and “fills in the gaps.”  This repair mechanism is frequently in use because it is used to repair damage from UV radiation, especially thymine dimers.  To accomplish this, the lesion is first detected by XPC and HR23.  Next, XPB and XPD, complexed with TFIIH, unwind the DNA via helicase activity.  Then the entire base and ribose-phosphate backbone is removed in a span of 24-32 nucleotides by XPC and ERCC1-XPF, which act as endonucleases.  DNA polymerase and ligase then replace the missing nucleotides and close the DNA.

NER simplified
  1. Detection of lesion by XPC and HR23
  2. XPB, XPD, and TFIIH bind and act as helicases
  3. XPC and ERCC1-XPF act as endonucleases to remove 24-32 nucleotides
  4. Replacement and ligation of DNA by DNA pol and ligase
An additional pathway involves the binding of CSA and CSB to the lesion when RNA polymerase II is actively transcribing the DNA into mRNA.  CSA and CSB then recruit the same proteins as described above and help in DNA repair. 

Diseases associated with defects in these two pathways can have severe phenotypes.  Xeroderma pigmentosum manifests itself as extreme sensitivity to UV light with an increased risk of skin cancer.  Seven genes have been found to be involved in the disease phenotype and can be associated with different severities of the disease.  An excellent review of XP can be found here. Additionally, Cockayne syndrome is associated with defects in the CSA/CSB pathway.  More information about this disease can be found here.

Similar to nucleotide excision repair, base excision repair fixes small lesions in DNA, but instead of using endonucleases to cleave small sequences of DNA, base excision repair removes single bases, not the entire nucleotide.  The incorrect base often takes the form of uracil, which can be accidentally incorporated in place of dTTP or via the deamination of cytosine.  While the incorporation of dUTP instead of dTTP is not mutagenic (A will still bind either T or U), deamination of cytosine can lead to a conversion of a C-G pair to a T-A pair. 

To repair via base excision repair, DNA glycosylases flip the incorrect uracil base to stick outside the DNA backbone.  AP endonuclease (APE) then forms a nick at the site of the lesion and removes the sugar phosphate (via lyase).  The missing base is then replaced by DNA polymerase β and the nick is sealed by DNA ligase.  The two DNA glycosylases (UDG: uracil DNA glycosylases) of mammals follow the replication fork and travel the DNA to find incorrect uracil bases.

Base excision repair simplified
  1. Uracil DNA glycosylase (UDG) recognizes uracil and flips the base
  2. AP endonuclease (APE) nicks the site of the uracil
  3. Lyase removes the sugar phosphate
  4. Replacement of base by DNA polymerase β
  5. Ligation of DNA by ligase

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