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