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Repair Mechanisms
- Direct repair
- Mismatch repair
- Base excision repair
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MMR
Methylation-directed mismatch repair
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How to repair structured problems
- direct repair
- mismatch repair
- excision repair
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DNA glycosylases
•several structural classes
•not necessarily linked to mechanism
- A)T4 endonuclease V w/ T-T
- B)U DNA glycosylase w/ pseudo-U
- C)8-oxoguanidine glycosylase w/ 8-OG
- D)alkyladenine glycosylase w/ ethenoadenine
- E)MutM w/ 8-OG
- some bind metal - not catalytic
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AP endonuclease generates _____
nick
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____ and ____ complete repair
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Nucleotide Excision repair pathway
- Add radioactive deoxythymidine triphosphate (dTTP) to media – cells take up in S phase
- But upon UV treatment, cells also take up outside of S phase
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Nucleotide excision repair (NER)
- 1.Targets bulky lesions that distort helix
- 2.Steps carried out by uvr gene products
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Causes of Double Strand Break Formation
- •Irradiation, UV rays, certain chemicals, oxidative damage
- •Base/nucleotide damage→Collapsed fork
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Why are DS breaks bad?
- •Inhibit replication/transcription
- •Can lead to chromosomal rearrangements
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Translesion synthesis (bypass repair) by TLS pol’s
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Repair of breaks
- 1.Recombinational
- 2. Non-homologous end joining
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Homologous Recombination
- Requires a “good” DNA strand to act as a template to repair “bad”
- Involves a recombinase – enzyme capable of matching “good” and “bad” DNA in a sequence dependent manner
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How to resolve:
- 1.Synthesis-dependent strand annealing (SDSA)
- 2. DSBR
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How does the recombinase work?
- Binds to SS DNA on “bad” strand
- Binds to DS DNA on “good” strand
- Aligns the “bad” strand with the complementary strand on the DS DNA – displacing the identical “good” strand
- Now the gap from the DS break is filled in using the “good” strand as the template
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Compare/Contrast HR (homologous recombination) and NHEJ (non-homologous end joining)
- •Similarities:
- •Type of DNA damage repaired
- •Both can result in mutation of DNA – discuss the mechanism
- •Differences:
- •Genomic complexity determines the balance of use
- •When? Cell cycle
- •Req for homologous DNA
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How to get a Knock-Out Cassette (or any foreign DNA) into a cell - Transformation
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