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When is NER used?
Removal of bulky adducts
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What governs NER efficiency?
Helical distortion (by the adduct)
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Types of NER
- Short patch - 12-13nt
- Long patch - 2kb
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Complex used in Short patch NER (prokaryotes)
UvrABC endonuclease
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UvrABC structure
- Tetramer
- UvrA dimer
- UvrB dimer
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UvrAB
- Scans DNA for distortions
- UvrA bends the helix and dissociates
- UvrB melts the helix (helicase)
- UvrA recruits UvrC
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UvrBC complex
- Incises 4-5nt to the 3' side of the lesion
- Then incises 8nt to the 5' side of the lesion
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UvrD
Helicase, removes the fragment produced by UvrBC
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Enzyme(s) responsible for filling in the gap causes by UvrBC (prokaryotes)
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Robert Painter
- Discovered eukaryote NER (without knowing it)
- Noted DNA synthesis not occurring in S-phase (after UV exposure)
- Unscheduled DNA synthesis
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Audioradiographic assay
- Used by Robert Painter
- Measured incorporation of [H3]deoxythymidine triphosphate in S-phase
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Xeroderma pigmentosum
- Autosomal recessive
- Increases susceptibility to skin cancer
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Variants of xeroderma pigmentosum
XPA, XPG, XPV
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James cleaver
- Studied patients with Xeroderma pigmentosum
- Determined that NER was not present in these patients
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Dirk Bootsma
- Discovered genes associated with Xeroderma pigmentosum
- Fused different XP variant cells
- Complementation meant cells had different defective genes
- Non change meant the defective gene was the same in both cells
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DNA Damage binding complex proteins (Eukaryotes)
- XPE (aka DDB2)
- DBB1
- XPC
- RAD23B
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First two proteins of the DNA Damage binding protein
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What is TFIIH
- A transcription factor, melts the DNA
- Part of the DNA Damage binding protein
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What is the role of XPG
To stabilize the DNA Damage binding protein with the DNA
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TFIIH Subunits
- XPB, 3'-5' helicase
- XPD, 5'-3' helicase
- 4 others
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How is the helix opened? (eukaryotes)
TFII opens ~30nt around the lesion (uses ATP)
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Proteins that work on the repair bubble (eukaryote)
ERCC1/XPF (Dimer)
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Proteins of the preincision complex
- XPA
- XPG
- TFII H
- ERCC1/XPF
- Maybe more..
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Incisions made by the preincision complex
- 22-24nt in the 5' direction (ERCC1/XPF)
- 5-6nt in the 3' direction (XPG)
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Role of XPA and RPA
Stabilize melted DNA (like SSBs)
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Exonuclease
Remove nucleotides from the end of the polymer
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Endonuclease
Remove nucleotides from the middle of the polymer
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Proteins of the NER repairosome with endonuclease activity
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Final proteins of eukaryote NER
- RFC (loader), PCNA, polymerase delta or epsilon
- DNA ligase I
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TCR and GGR
- Transcription coupled repair (NER) - Similar to GGR, except the polymerase initiates the process
- Global genome repair (NER)
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Proteins not required in TCR NER
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How does the polymerase detect damage in TCR
- It pauses on the damaged base
- Frequent pausing can trigger apoptosis
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TCR is NER exclusive (T/F)
F, TCR is used for BER too
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Effect of TCR on rates of CPD repair
80% CPDs removed from transcribed strands whithin 2hrs, compared with 10% for GGR
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TCNER mechanism
- The RNA pol II stalls
- CSA and CSB are recruited
- The pol II is removed and degraded
- NER proceeds
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