Hank Fenton, DNA damage, and why we store our DNA in EDTA

Jan 17 2011 Published by under Uncategorized

So chatting with a undergrad that was shadowing me last week before my surgery we got on the topic of DNA damage and that a primary endogenous source of DNA damage comes in the form of hydrolytic damage causing loss of bases, as well as base modification (18,000 events per cell per day).  Similarly another major cause of damage created in the cell is reactive oxygen species that arise as a result of mitochondrial leakage (3-4,000 events per cell per day).

What usually occurs is that as a result of mitochondrial metabolism molecular oxygen is converted to superoxide (·O2-).  This ROS species is not particularly dangerous to DNA because it is short-lived and gets rapidly converted to peroxide by superoxide dismutase (H2O2).  Peroxide is what poses a huge threat to DNA because when peroxide is in close proximity with our DNA it gets converted to hydroxyl radical (·OH) through what is called the Fenton Reaction.

Fe2+ +  H2O2 -->  Fe3+ +  ·OH  +  -OH

British chemist Hank Fenton discovered this reaction way way back in 1894 but I’m going to assume he had no appreciation for how potent a genotoxic insult this simple reaction would play in our bodies.   The key part of this reaction lies in ferrous iron (Fe2+) found in the DNA that plays a key role in the conversion of peroxide to hydroxyl radical.

A common form of oxidative DNA damage is the conversion of guanine to 8-oxo-guanine, which while in the syn conformation, can mispair with adenine.  The end game is that the damage has resulted in a mutation, but fear not there are specialized enzymes called DNA glycosylases that can remove this damage base and incorporate the proper nucleotide triphosphate.  If you are interested this pathway is called base excision repair and the particular DNA glycosylase that is predominantly responsible for the removal of 8-oxo-guanines from our genome is called OGG1.

So lets end this post on why you toss in some EDTA in addition to Tris when resuspending DNA.  Its because we use it to chelate these metal ions like iron so that it cannot participate in the Fenton reaction and cause damage to our precious DNA samples in addition to binding other metal ions required for enzymatic activity by nucleases, which can further degrade your sample.

Postscript:  One last note on DNA storage, which we debate back and forth endlessly with each other.  Most of us store DNA at 4 °C for short-term use, but keep most of our precious samples at -80 °C for long-term storage.  You can store DNA at -20 °C but you run the risk of it falling out of solution but vortexing will easily bring it back into solution.  Note this is fine for plasmids, but vortexing will rip the shit out of genomic DNA so be kind to it and store it at -80 °C.  And one last bit, don’t store DNA in a freezer model that automatically defrosts, this freeze thaw cycle will end up degrading your DNA.

Values of DNA damage come from:  PMID  12760027

21 responses so far

  • Jade says:

    Excellent post!! Thanks!

  • pd says:

    Interesting stuff, I've been wondering about the EDTA. Thanks!

  • Dr Becca says:

    GR--this is super interesting, but for the love of god, would it kill you to use a comma once in a while? I say this with nothing but the utmost affection.

  • UnlikelyGrad says:

    I work with reactive oxygen species like superoxide and hydrogen peroxide. When I first started this project, I thought, "How dumb. Why would anyone care about these things?" The more I read, the more I am amazed at how ubiquitous they are.

    BTW, most cells contain fairly high levels of catalase or other peroxidases to decompose hydrogen peroxide. When you lyse a cell, these get spilled out with all the other cell guts (which does interesting things to water chemistry...I work in the aquatic systems). However, I assume these get removed along with all of the other proteins when you extract DNA. I'd never thought about that before.

  • SB says:

    Fascinating post, GR. One of my profs thinks that freeze-thawing causes the formation of the reactive oxygen species, which lead to DNA damage... but if the reaction is enzymatic as you suggest, I can't see why the freeze-thaw cycles would be an issue; if anything, I would think they would kill those enzymes!

    Interesting note about the EDTA. I always resuspend my DNA in water and it's worked fine for now but I'll keep that in mind!

    • Genomic Repairman says:

      Sb, DNA is a surprisingly resilient polymer but water itself causes damage to DNA in the form of base hydrolysis. Going from aqueous to solid state and back wreaks havoc on DNA structure causing the DNA to become brittle and fragment.

  • Vortexing makes surprisingly little difference to the size of genomic DNA fragments in DNA preps. If you're reasonably gentle with the DNA (no vortexing, just some gentle pipetting up and down) the fragments are mostly between 50 and 200 kb. If you vortex the hell out of it for a minute, the fragments are still mostly in the 50-100 kb range. If you want to get it any smaller you need some fancier technology (sonicator, nebulizer...) or a well-controlled nuclease.

    Thanks for the info about the Fenton reaction - I always thought the EDTA was just to chelate magnesium away from those pesky nucleases!

  • Back in my grad school days, I did a massive genomic DNA prep (can't remember the producer of the kit) of my target organism which sat on my benchtop, stored in TE, for my entire graduate school career. Used it for all of my PCR reactions, and it worked up until the day I threw it out (the day after my defense, approximately 4.5 years later).

    Maybe I played fast and loose with the DNA gods, but as long as the Tris is there to buffer your solution, and the EDTA is there to chelate your ions, you won't get biological or chemical hydrolysis.

  • BrooksPhD says:

    nice post mate. I'm with Rosie on thinking it was just to make my PCR easier!

  • becca says:

    Anyone have any numbers on the % of damage due to nucleases vs. peroxide damage in different types of preps of DNA? Does the concentration affect this?

  • Genomic Repairman says:

    Becca, just tooling around to look for a second, I couldn't find anything. I don't know if anyone has done head to heads for nuclease vs peroxide because there are different read outs for damage.

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