8/1/01

Lecture #14 - DNA repair - part II

Recombination repair

• This is also called retrieval or post-replication repair because it cannot function unless the cell has already replicated its DNA.
• This type of repair requires a recombination event that is dependent on the recA gene. recA encodes a recombinase enzyme.
• The actual DNA damage is not removed &endash; it is more like a temporary measure to keep the cells alive until the next generation when the DNA can be repaired by other mechanisms like nucleotide excision repair (NER)
• It is usually utilized to repair bulky distortions like thymine dimers or alkylation events.
• Recall that DNA polymerase cannot replicate through thymine dimers; instead it skips over the dimer, leaving a gap in the opposing strand DNA.
• The recA enzyme then excises the corresponding piece from the good copy of the DNA and inserts it across from the dimer on the damaged copy. The good copy has no thymine dimer, so it can then fill in its new gap with traditional DNA synthesis methods. See Fig. 14.31.
• Evidence for the necessity of recombination repair comes from looking at ability of wild type E. coli and mutants to repair DNA damaged by uv light. Wild-type cells show an inverse linear relationship between uv dose and cell viability. uvrA^- mutants which cannot use NER show a higher mortality, and recA mutants are still higher. The uvrA^-/recA^- double mutant is most striking &endash; it can barely survive with any exposure to uv light. (Note this experiment takes place in the dark to avoid exposure to the 370 nm light that would activate the phr photoreactivation repair enzyme.)

Transcription-coupled repair

• Evidence for this type of repair comes from observations that actively transcribed DNA is repaired more efficiently than non-transcribed DNA.
• This is a strand-specific repair. The template strand is repaired while the coding strand is not.
• This method is present in both bacteria and eukaryotes.

Transcription coupled DNA reapair in bacteria:

• A protein called Mfd recognizes DNA damage and is interestingly somehow associated with RNAP. When the Mfd recognizes the damage, it recruits the NER machinery (uvrA-D) - this causes the RNAP to fall off of the template. The NER machinery then repairs the damage. The next time transcription is attempted, the damage will have been fixed. See Fig. 20.14.

Transcription coupled DNA reapair in eukaryotes:

• In eukaryotes the mechanism is more sophisticated, a difference which may be due their gene structure. Recall that eukaryotes often have large introns, so the primary transcript can be very large. For example, the human dystrophin gene (when mutated causes muscular dystrophy) is 2.5 x 10⁶ bases. compare with the entire E.coli genome which is 4 x 10⁶ base pairs.
• Suppose the RNAP encountered a thymine dimer in the final hundred bases of transcription of the dystrophin gene. It would be an extremely disadvantageous if RNAP had to dissociate from the template and re-transcribe the entire gene. This would waste a lot of energy.
• For this reason, transcription-coupled repair in eukaryotes does not involve the RNA Pol II dissociating from the DNA.
• The overall process is similar to that in bacteria. First the damage is recognized, then the RNA Pol II pauses or backs up a bit. The DNA repair machinery is recruited, the error is fixed, and the RNA Pol II is back on its way. See Fig. 20.15.
• The DNA repair activity (or at least part of it) is actually a component of the RNA Pol II basal transcription machinery. It is present in TFIIH.

TFIIH has several activities:

• 1) ATPase
• 2) helicase
• 3) kinase (to phosphorylate CTD)
• 4)DNA repair.

Nucleotide excision repair in mammals

• Work on mammalian NER came out of the study of a rare recessive disease called xeroderma pigmentosa. Patients with this disease are hypersensitive to sunlight, suffering skin lesions and 1000 times the normal risk of skin cancer (risk for other cancer remains normal.)
• Diseased cells were cultured, exposed to uv light and assayed for NER. They were found to have no NER activity at all.
• It turns out that nine separate genes are involved in the NER pathway and a mutation in any one of them can cause the XP disease.
• The components of the NER pathway in mammals are not homologous to those in E.coli, but the components function similarly.

• XPC plays a major role in damage recognition while XPA plays only a minor role.
• XPB and XPD are helicases &endash; they are the componenets of TFIIH that are responsible for its helicase activity.
• RPA is a single stranded binding protein &endash; it coats the unwound single stranded DNA.

• Recall that E.coli has multiple mechanisms to remove thymine dimers (Phr, NER, recombination repair). Mammals seem to only have the NER mechanism wherease bacteria, plants and reptiles all have multiple methods. Did other organisms gain these other methods or did mammals lose all except NER?
• It makes sense from an evolutionary perspective that we lost the backup repair pathways. When mammals began, they were furry (best part of the lecture), lived underground, and were nocturnal. Therefore they didn't spend a lot of time in the sun, so there was no selective pressure to maintain the DNA repair mechanisms designed to correct DNA damage due to uv light exposure.

Human diseases associated with mutations in TFIIH

These are all caused by mutations of the XPB and XPD components of TFIIH.

• 1) XP (see above)
• 2) Cockaynes Syndrome &endash; patients are dwarfs and mentally retarded but show no increase in skin cancer. these cells are actually defective not in NER but in transcription-coupled repair.
• 3) Trichothiodystrophy (TTD) &endash; patients have brittle hair, are mentally retarded, have fish-like scales on their skin, and are sun-sensitive but show no increase in skin cancer. Molecular defect is in transcription.

So XPB and XPD have three functions: 1) basal transcription 2) NER 3) transcription-coupled repair.

• XP reflects a problem in #2 but normal #1 and #3.
• TTD is a defect in #1 with normal #2 and #3.
• Cockaynes syndrome is a problem with #3 with normal #1 and #2.

Mismatch repair in mammals

• Defects in mismatch repair in humans are associated with a certain type of inherited colorectal cancer which accounts for 5% of all colon cancers.
• This form of colon cancer is inherited as an autosomal dominant trait although the mutations themselves are recessive (this type of inhertance is often associated with tumor-suppressor genes).
• Patients are heterozygous for the gene at birth, but they tend to acquire mutations in the good copy during life, rendering them homozygous for the mutant and thus suceptible to the disease.
• A number of genes can cause this &endash; they are mutS homologs (MSH 2, 3 and 6) and mutL homologs (MLH1, PMS1 and PMS2).
• Researchers attempted to knock out these genes in mice to see if they could develop a good colon cancer model. When they knocked out PMS2 and MSH2 the mice got cancer, but the wrong kind. They developed lymphomas (tumors of the immune system) and sarcomas (variable tumors) instead of colon cancer.
• So the effect of these genes appears to be species-specific; the difference in mouse/human lifespan may account for this.
• These mutant mice were found to be defective in mismatch repair.
• They also displayed microsatellite instability &endash; repetive genome sequences were not replicated/recombined correctly.
• This caused defects in chromosome synapsis during meiosis so the mice could not perform recombination. All mutant males were in fact sterile for this reason.
• This suggest that the mismatch repair machinery is also involved recombination. Mismatch repair machinery in mammals may prevent similar but not identical sequences from recombining incorrectly.

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