Note: Measure DNA repair_2014
Note: Measure DNA repair_2009
doi: 10.1080/13102818.2009.10817632
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DNA mutations can cause diseases - thus understanding how cell repairs DNA is important.
The ability to repair DNA to return to homeostasis state;
Repair capacity
Repair rate
Repair specificity
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Content
Base sequences are kept securely by the backbone of phosphodiester bonds and N-glycosidic bonds -- very stable at the physiological condition
Base stacking -- save the base from exposing to environment
Any modification of the chemical structure of DNA -- DNA damages
DNA damage
Halt transcription
Cause mutation during replication
SSB
Phosphodiester bond is being hydrolyzed -- leaving 5’ end phosphorylated and 3’ end deoxyribosyl moiety with 3’OH group
SSB forms from the digestion of endonuclease
Damaged base must be removed from phosphodiester backbone -- SSB forms
Pyrimidine dimers
UV light (260 nM) -- DNA expose -- causing photoproducts
Two major types of photoproducts
Cyclobutane pyrimidine (T,C,U) dimers
Not significantly causing conformational distortion of DNA double helix
Pyrimidine-pyrimidone phtoproducts
Causing a major distortion of DNA double helix
- Thus, two adduct could be differentiated through its lability to akali which cyclobutane dimers insensitive
Modification and adducts
Small one
Oxidative condition within the cell
Purine -- 8-oxopurines
Pyrimidine -- opening imidazole ring -- formamidopyrimidine
Large one
Bulky adduct -- found in some chemical modification like cisplatin (alkylating agents)
Mismatches
Usually occur during the course of DNA replication
Double strand breaks
By definition interstrand cross-links
Super lethal due to the lack of template strand to repair
Many SSBs can cause DSB by mean of close proximity
ICL
Cisplatin, mitomycin C
The lesion is complex to repair -- require more than 1 pathway to repair
DNA repair pathways
Reversal of damage
Base excision repair
Nucleotide excision repair
Mismatch repair
NHEJ
HR
Methods to measure DNA repair
How to measure repair rate;
Monitoring the removal of DNA damage
Monitoring the restoration of the activity of the damaged DNA
Pyrimidine dimer
Using specific enzyme called T4 endonuclease
Alkaline electrophoresis
Observe the band pattern on gel electrophoresis, more ladders -- more fragments -- more defect in DNA repair
Alkaline elution
Measuring SSB
Must contain high numbers of breaks in order to be visualized by electrophoresis techniques
Used to monitor and compare repair kinetics of different normal and malignant cells -- it also has automated system
Agarose gel electrophoresis
Applying to detecting DSB
Pulse field gel electrophoresis is used to detect the repair kinetics of random DSBs
ICL could be detected in this way through the round of denaturation/renaturation -- ICL containing lesion -- has slower kinetic thus move through agarose at slower rate
Source: Protecting the aging genome
Comet assay
Single cell gel electrophoresis
Applying to detect the effect of a broad variety of agents
Two condition used;
Neutral comet assay
Measure only DSBs
Alkaline comet assay
Measure both SSBs and DSBs
Amount of DNA tail -- indicative for the number of breaks -- thus could be used to monitor repair capability
ICL will be analyzed in reverted way
Treat cell with ICL
Generating break on DNA
Run on alkaline agarose
ICL-adducted DNA -- remain in the hole, thus no tail, reflecting no ICL repair activity
Assay is sensitive, thus can be used for low number of breaks
Unscheduled DNA synthesis
Mean non-replicative DNA synthesis
Considering based on DNA damage -- Remove damage -- DNA synthesis to fill gap
Quantitative tool to measure DNA repair both in vivo and in vitro
Measure through the incorporation of labeled nucleotides under conditions of inhibitor (ex. hydroxyurea) of replicative DNA synthesis
Repair rates can be determined in different regions
PCR assay
Relying on the fact that the DNA lesion blocks DNA polymerase during DNA synthesis -- thus DNA-containing lesion template could not yield any PCR products.
Determined DNA repair of UV/chemically produced DNA lesion
Assaying DNA repair rates in cancer cells of different rat tissues and to compare repair rates of different DNA lesion
Mass spectrometry
Most direct and specific method to determine different type of DNA modification
To quantify -- require the std.
We harvest cell at different time points and detect the lesion with mass spec
Obstacle; finding the condition to hydrolyse DNA without changing the chemical nature of the products
Immunno assay
Two approaches
Ab raise against based modification
Hard to produce antibody
Some modified bases are not antigenic
- Protein involved in DNA repair
- relying on the fact that protein aggregate and assemble at site of damage and disassemble after damage is repaired
- H2AX detecting DSB, Rad51 detecting HR, PCNA detecting NER
- Sensitive method 1 DSB in 1 single cell could be seen by this method
Cell free protein extracts
Measuring repair efficiencies of different cells and tissue
Using plasmid with targeted lesion as the template to detect repair efficiency in cell free system
The repair efficiency could be measure through
PCR
Transformation efficiency, if the template contains antibiotic resistant
Host cell reactivation assay
Measuring the repair that uses restoration of transcription and expression of damaged DNA
Using exogenous DNA containing a reporter gene is damaged in vitro -- transfect into host cells -- measure through the activity of reporter gene
Used with higher eukaryotic cells due to they can be transfected with high efficiency
Report on quantifying DNA repair capacity in studies of lung cancer, skin, head and neck, prostate cancers -- after treatment with genotoxic agents.
HCR -- assay all types of DNA damages
Conclusion
Why measuring DNA repair rate and capacity
It could be used as biomarkers to initiate the treatment, predict the resistant, and use as prognostic value for risk assessment upon exposing to harmful agents
Low in mismatch
Risk for colon cancer
Low in NER
Risk for skin cancer
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