Often for the DNA glycosylase enzyme assay we can see that the DNA glycosylase is incubated with oligonucleotide in 37C and then reaction is stopped by addition of formamide gel loading buffer (80% formamide/1 mM EDTA, pH 8.0y0.1% bromophenol blue/0.1% xylene cyanol). How the reaction is stopped by this buffer?

  • $\begingroup$ Do you have a reference for that? $\endgroup$ – canadianer Jul 19 '14 at 19:34
  • $\begingroup$ Cloning and Characterization of a Functional Human Homolog of Escherichia coli Endonuclease III Aspinwall, Richard ; Rothwell, Dominic G. ; Roldan-Arjona, Teresa ; Anselmino, Catherine ; Ward, Christopher J. ; Cheadle, Jeremy P. ; Sampson, Julian R. ; Lindahl, Tomas ; Harris, Peter C. ; Hickson, Ian D. Proceedings of the National Academy of Sciences of the United States of America, 1997, Vol.94(1), pp.109-114 [Peer Reviewed Journal] $\endgroup$ – Piesel Jul 19 '14 at 22:26
  • $\begingroup$ Formamide is a common loading buffer for denaturing gel electrophoresis of nucleic acids. I'd assume that it also denatures most enzymes and stops the reaction that way. $\endgroup$ – Mad Scientist Jul 20 '14 at 12:32

Escherichia coli endonuclease III (which I will now refer to as Nth), and the human homolog hNTH1, has both glycosylase and AP lyase activity. The section of the article you're referring is describing an assay of lyase activity. The assay tested both Nth and hNTH1 as well as HAP1, a human AP hydrolase that cleaves 5' to the AP site. Nth and hNTH1 were found by this assay to cleave 3' to the AP site. HAP1 is dependent on $Mg^{2+}$ for catalytic activity. hNTH1 (and likely Nth, given the high conservation) are dependent on $Mg^{2+}$ for substrate specificity (Eide et al., 2001). hNTH1 and Nth contain an $Fe-S$ cluster that is involved in DNA binding (Thayer et al., 1995).

Originally I thought that the reaction is stopped with EDTA. EDTA is a chelating agent; it binds and sequesters metal ions and prevents their use by enzymes. EDTA would prevent catalysis by HAP1 and DNA binding by Nth/hTNH1.

However, Chris pointed out that the loading buffer contains 1 mM EDTA while the reaction buffer contains 5 mM $MgCl_2$. While the paper doesn't give the volumes of reaction and loading buffer mixed together, it is perhaps unlikely that EDTA chelation of $Mg^{2+}$ is the sole mechanism for stopping the reaction. The high concentration of formamide (80%) in the loading buffer could then be responsible. Formamide is commonly used to denature nucleic acids and, at high concentrations, would likely have an effect on enzyme function.


Eide L, Luna L, Gustad EC, Henderson PT, Essigmann JM, Demple B, Seeberg E. 2001. Human Endonuclease III Acts Preferentially on DNA Damage Opposite Guanine Residues in DNA. Biochemistry-US. 40(22):6653–6659

Thayer MM, Ahern H, Xing D, Cunningham RP, Tainer JA. 1995. Novel DNA binding motifs in the DNA repair enzyme endonuclease III crystal structure. EMBO J. 14(16):4108–4120.

  • $\begingroup$ How much magnesium is present in the buffer? The EDTA in the buffer is only 1mM, so only 1mM of magnesium is complexed. I doubt that this is enough. Additionally, this buffer is not only TE but contains 80% Formamide. $\endgroup$ – Chris Jul 20 '14 at 7:56
  • $\begingroup$ @Chris: Thanks, that's a good point. The reaction buffer contains 5 mM &Mg^{2+}$. However, they don't give the volumes of reaction and loading buffer that are added to each other and electrophoresed. Do you think that formamide is the major factor stopping the reaction? I assumed it was there to denature the DNA. Does it also denature enzymes? Or perhaps the enzymes don't function on ssDNA. $\endgroup$ – canadianer Jul 20 '14 at 17:36
  • $\begingroup$ I think so. I haven't checked if formamide reacts with proteins. That would be one possibily. $\endgroup$ – Chris Jul 20 '14 at 18:21
  • $\begingroup$ I've edited my answer, though I don't feel it answers the question anymore. I wish I could find some literature about it. $\endgroup$ – canadianer Jul 20 '14 at 20:02
  • $\begingroup$ Thank you very much for your help. As you mentioned in your response, AP andonucleases may incise 3’ and 5’ to the AP site. I read more about it and I am confused at some point. I have found: There are four possible incision sites for AP endonucleases, and the enzymes have been classified according to their sites of incision: class I and class II AP endonucleases incise 3’ and 5’, respectively, to the AP site, both leaving 3’-OH and 5’-P termini. If for class II AP endonucleases incision is at 5' to the AP site, how it is that there is generated 3'-OH and 5'P? $\endgroup$ – Piesel Jul 20 '14 at 23:00

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