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Excision Repair

Notes

Excision Repair

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EXCISION REPAIR MECHANISMS

Mismatch-repair

• Fixes replication errors missed by DNA Pol proofreading (cannot repair damage)

Base excision repair

• Damaged bases removed and replaced
• Deamination & depurination (most common spontaneous damages)
• Alkylation (except for O6-alkyl guanine)
• Oxidation

Nucleotide excision repair

• Damaged nucleotides removed and replaced
• 2 types: global genomic NER (transcriptionally inactive) & transcription-coupled
• Repairs wider variety of lesions that distort DNA helix
• Pyrimidine dimers and other intrastrand adducts
• Bulky group addition (via carcinogen-exposure)

BASE EXCISION REPAIR
• Model: cytosine spontaneously deaminates to uracil

  1. DNA glycosylase excises uracil: cleaves N-glycosidic bond between base & deoxyribose
    • Produces apyrimidinic site (AP site)
  2. AP endonuclease cleaves deoxyribose backbone adjacent to AP site
    • Excises deoxyribose moiety
  3. DNA polymerase and ligase fill/seal gap with cytosine
    • Prokaryotes: DNA Pol. I
    • Eukaryotes: DNA Pol. beta

NUCLEOTIDE EXCISION REPAIR
• Model: thymine dimer in E. Coli (prokaryotes)

Global genomic NER: transcriptionally inactive region

• Uvr genes: discovered in E. Coli, mutations conferred extreme UV light sensitivity

  1. UvrA recognizes helical distortion as damage
  2. UvrA recruits uvrB and uvrC

Intertextual variation exists for above step.

  1. Uvr B and C cleave DNA on either side of damage
    • Uvr ABC complex: excinuclease
  2. Helicase unwinds DNA and releases damaged segment
  3. DNA polymerase fills gap and ligase seals it
    • DNA Pol. fills gap in 5' to 3' direction
    • Prokaryotes: DNA Pol. I
    • Eukaryotes: DNA Pol. delta and epsilon

Transcription-coupled NER: transcriptionally active region

• Repairs more rapidly than global genomic pathway

  1. NER enzymes recognize RNA polymerase stalled by lesion and displace it
    • E. Coli: NER enzymes are Mfd proteins
    • Humans: RNA polymerase II (prokaryotes only have one RNA Pol.) & CS proteins
  2. Mfd proteins (E. Coli) and CS proteins (humans) recruit other proteins to site
    • E. Coli: Mfd proteins recruit uvr proteins
    • Humans: CS proteins recruit more CS and XP proteins (amongst others)
  3. Recruited proteins cleave and excise damaged oligomer
  4. Identical to Step 5 in global genomic pathway

CLINICAL CORRELATION

Xeroderma pigmentosum (XP)

• Rare genetic defect produces dysfunctional XP proteins
• Patients extremely sensitive to UV light: develop skin cancers in sun-exposed areas

Full-Length Text

  • Here we will learn about DNA excision repair, in which damaged or mismatched DNA bases or nucleotides are removed and replaced.
  • To begin, start a table to learn the key types of excision repair.
  • Denote that they are:
    • Base-excision repair, in which damaged bases are removed and replaced.
    • Nucleotide excision repair, in which entire damaged nucleotides are replaced.
    • Mismatch repair, which is a unique pathway that fixes mismatched bases incorrectly incorporated during DNA replication. (We cover mismatch repair in detail, elsewhere.)
  • Next, denote that there are two types of nucleotide excision repair.
    • The first is called global genomic nucleotide excision repair (NER), which repairs damage in a transcriptionally inactive region of DNA.
    • The second is transcription-coupled NER, which repairs damages in a transcriptionally active region.

We learn each of these mechanisms.

Let's start with base-excision repair.

  • First, write that this pathway addresses the following forms of DNA damage:
    • Deamination or depurination, which are the two most common forms of spontaneous damage.
    • Alkylation (except for O6-alkyl guanine).
    • Oxidation.

We will use deamination as our model for this pathway.

  • To begin, draw a segment of double stranded DNA and label the 5 prime and 3 prime ends of each strand.
  • Attach cytosine to the 3 prime to 5 prime strand.
  • Attach guanine (its complement) to the opposite strand.
  • Next, indicate that cytosine gets spontaneously deaminated; deamination is one of the most common forms of spontaneous DNA damage.
  • Now, draw the product of this spontaneous deamination: cytosine becomes uracil.

Next, let's illustrate base-excision.

  • Step 1: An enzyme called DNA glycosylase excises uracil; specifically, it cleaves the N-glycosidic bond between the base and the deoxyribose backbone.
  • Now, draw the product and label the apyrimidinic site (AP site).
    • If hypoxanthine, the product of adenine deamination, had been excised instead of uracil, this would be an apurinic site (still AP site).
  • Step 2: AP endonuclease cleaves the deoxyribose backbone adjacent to the AP site, excising the deoxyribose moiety.
  • Now, draw this product.
  • Step 3, the final step: DNA polymerase and ligase fill the gap with the appropriate nucleotide (cytosine), and ligase seals it in place.
  • Indicate that this is specifically DNA polymerase I in prokaryotes and beta in eukaryotes.

Now, for nucleotide excision repair.

  • Write that unlike base excision repair, it can repair a wide variety of lesions that distort the DNA helix and is the only repair pathway that can address:
    • UV-induced pyrimidine dimers and other intrastrand adducts.
    • Bulky groups, which are often added to nucleotides upon exposure to carcinogens.

Now, let's illustrate this pathway.

  • Draw a segment of double stranded DNA with a large bend in each strand.
  • Label the 5 prime and 3 prime ends.

Now, let's illustrate the DNA damage on the 5 prime to 3 prime oriented strand.

  • Show that two adjacent thymine nucleotides form a dimer.
  • Show that the corresponding adenine nucleotides on the opposite strand cannot hydrogen bond with the thymines.

Now, for repair.

  • Start with global genomic NER, which repairs transcriptionally inactive regions.
    • We will use prokaryotes, specifically E. coli, as our model and point out key differences in the mammalian pathway.
  • First write that NER enzymes derive from Uvr genes in E. Coli and XP genes in humans.
  • As a side note, write that the Uvr genes were discovered in E. Coli because mutations at these loci conferred extreme UV light sensitivity.
  • Step 1: UvrA recognizes helical distortions in DNA as damage.
  • Step 2: UvrA recruits uvrB and uvrC to the site of damage.
    • Intertextual variation exists regarding this step.
    • Some texts outline UvrA recruiting uvrB, which then recruits uvrC.
    • Other texts describe uvrA release once uvrB and uvrC are recruited.
  • Step 3: Uvr B and C cleave DNA on either side of the damage.
  • Illustrate this by redrawing our damaged DNA with nicks on the 3 prime and 5 prime sides of the thymine dimer.
  • Write that the Uvr ABC complex is known as an excinuclease; it recognizes and cleaves the damage.
  • Now, step 4: helicase unwinds the DNA and releases the damaged segment. In prokaryotes, this helicase is actually Uvr D, another Uvr gene protein.
  • Draw the product of this step: two DNA segments with a large gap in one strand.
  • Finally step 5: DNA polymerase fills the gap and ligase seals the ends.
  • Show that it synthesizes DNA in the 5 prime to 3 prime direction.
  • Indicate that this is DNA polymerase I in prokaryotes and DNA polymerase delta and epsilon in humans.

Finally, let's learn transcription-coupled NER, which addresses the same scope of DNA damage as global genomic NER, but does so within actively transcribed genes, which are repaired more rapidly than in transcriptionally inactive DNA.

  • Step 1: NER enzymes recognize an RNA polymerase that is stalled by a lesion in DNA and displace it.
    • Write that this is RNA polymerase II in humans.
    • Prokaryotes only have one RNA polymerase.
    • Unlike the global genomic pathway, this step is not catalyzed by uvr or XP gene products. Instead write that it requires Mfd proteins in E. coli and CS proteins in humans.
  • Step 2: Mfd proteins in E. coli and CS proteins in humans recruit other NER enzymes to the site.
    • Mfd proteins recruit uvr proteins.
    • Whereas CS proteins recruit more CS and XP proteins (amongst others).
  • For Step 3, indicate that these proteins cleave and excise the damaged oligomer.
  • Show that Step 4 (the final step) is identical to the last step in global genomic NER:
    • DNA polymerase fills the gap and ligase seals the end, using the same DNA polymerases as in the global genomic pathway.
  • As a clinical correlation, write that the xeroderma pigmentosum (XP) is a rare genetic defect that produces dysfunctional XP proteins making patients extremely sensitive to UV light and setting them up to develop skin cancers in sun-exposed areas.