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33 Cards in this Set
- Front
- Back
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what are the 2 mechanisms to excise and replace mismatched bases?
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1. EDITING PROOFREADING from DNA pol 3` exonuc activity
2. METHYL DIRECTED MISMATCH REPAIR (on same strand as mismatch) |
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What is dark repair? |
nucleotide excision repair transcription coupled repair |
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What is repair associated with replication |
editing CH3-directed mismatch repair |
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What does editing/proof reading repair do? |
corrected by excising mismatched base and replacing with correct base |
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Editing fixes the _______ made if DNA pol incorporates the ___________ ___________. What are the functions of DNA POL I and DNA POL III, and when they perform _________ to stop _______________, where does their 3` exonuclease activity come from?
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Editing fixes the _MUTATION__ made if DNA pol incorporates the __WRONG_ _NUCLEOTIDE___. What are the functions of DNA POL I and DNA POL III, and when they perform _EXCISION__ to stop _REPLICATION_, where does their 3` exonuclease activity come from?
FUNCTION. 3`EXONUC. MutD- mtnt of DNA POL I. REPAIR. PART OF ENZ. DnaQ -defective DNA POL III. REPLN. DnaQ. 3` exonuclease |
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What is the function of DNA polymerase I? |
REPAIR
3' exonuclease activity - part of the enzyme itself |
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What are steps of editing/proofreading repair? |
RECOGNITION - distortion of DNA helix by mismatched pair
EXCISION - 3' exonuclease stops replication until base excised
CORRECTION - replication continues, correct base inserted |
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What is the function of DNA polymerase III? |
REPLICATION
3' exonuclease activity - DnaQ |
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What are the characteristics of dnaQ / mutD mutants? |
- defective 3' exonuclease - much higher rates of spontaneous mutation - USED IN LABS |
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How is CH3-directed mismatch repair different? |
recognizes mismatches SOON AFTER replication - not at the 3' end - removes and replaces mismatch & DNA in same strand - gap is filled by DNA polymerase III |
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What system does CH3-Directed Repair use? What kind of methylation happens, and when, after replication? How does this work with the repair system? What kind of
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1. Q- DAM methylase - HEMI-METHYLATES newly replicated DNA (on the A in 5'-GATC-3')
- repair recognizes mismatches in HEMI-METHYLATED DNA -selectively excises UNMETHYLATED base = methyl directed dam- mutants = higher rates of mutation |
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How do MutS, MutL and MutH interact in CH3-directed mismatch repair? |
-MutS dimer recognizes DNA distortion and binds
-2 copies of MutL bind MutS
MutH binds this complex - activates endonuclease to cut nearest hemi-methylated sequence |
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What does UvrD helicase do in CH3-directed mismatch repair? |
Unwind DNA |
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What do ExoVII and RecJ do ? |
Degrade ssDNA 5' to 3' |
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What do ExoI and EcoX do? |
Degrade ssDNA 3' to 5' |
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What does CH3-directed mismatch repair? |
Many types of damage to DNA by MINOR distortions - mismatches - frameshifts - base analog incorporation - alkylation causing minor distortions |
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What is the genetic evidence for mismatch repair? |
- mut gene mutations have higher than normal mutation rates
- lambda phage heteroduplex experiments
-2-aminopurine sensitivity
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What are the 2 hypotheses for dark repair? |
Photoreactivation (light repair)
Dark repair / liquid-holding recovery |
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What is nucleotide excision repair? |
DNA containing damage is excised from DNA |
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What are the characteristics of nucleotide repair? |
* UV irradiation of bacteria * Hold in liquid buffer * Thymine dimers decrease * Thymine dimers appear in buffer |
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Nucleotide Excision Repair (NER) Step 1 |
UvrA2 + UvrB1 bind to DNA...goes along until finds lesion |
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NER Step 2 |
UvrB binds damage
UvrA replaced by UvrC |
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What do the lambda phage heteroduplex experiments show? |
* progeny phage show phenotype of parent grown in dam+ strain
* DNA repaired according to strand methylated |
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What is dark repair? |
- similar to photoreactivation
- UV-irradiated cells in dark before plating; higher survival |
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NER Step 3 |
UvrB cuts sugar-phosphate backbone
4-5 nucleotides (nt) away from 3' of damage |
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NER Step 5 |
UvrD helicase removes damaged oligonucleotide |
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NER Step 6 |
DNA pol I resynthesizes strand
uses 3'-OH as primer |
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NER Step 6 |
Ligase seals gap |
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What is transcription coupled repair (TCR)? |
Damaged DNA in transcribed genes FIXED FIRST
Prevents blocks in transcription & translation |
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How does TCR work? |
* RNAP encounters damage
* Backtracks
* Moves growing RNA into secondary channel |
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What is Mfd and how does it work? |
Mutation frequency decline OR transcription repair coupling factor (TRCF)
* binds DNA behind RNAP *pushes RNAP forward *if can't go because of damage, RNAP leaves *recruits UvrBCD nuclease to fix damage |
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NER Step 4 |
UvrC cuts sugar-phosphate backbone
7-8 nt away from 5' of damage
generates 3'-OH |
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what happens during recognition in editing\proofreading?
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helical distortion from the base pair mismatch is recognized
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