Action of Endo VIII - (May/03/2011 )

Hi everyone,

I am confused about the action of Endonuclease VIII. The NEB web site describes it as following:

Endonuclease VIII from E. coli acts as both an N-glycosylase and an AP-lyase. The N-glycosylase activity releases damaged pyrimidines from double-stranded DNA, generating an apurinic (AP site). The AP-lyase activity cleaves 3´ and 5´ to the AP site leaving a 5´ phosphate and a 3´ phosphate. Damaged bases recognized and removed by Endonuclease VIII include urea, 5, 6- dihydroxythymine, thymine glycol, 5-hydroxy-5- methylhydanton, uracil glycol, 6-hydroxy-5, 6-dihydrothymine and methyltartronylurea (1,2). While Endonuclease VIII is similar to Endonuclease III, Endonuclease VIII has β and δ lyase activity while Endonuclease III has β lyase activity.

Does this mean that the DNA won't be cleaved if there is a pyrimidine missing?

Should it be a apyrimidinic since it removes pyrimidines?

And I think it removes bad pyrimidines so, if you dont have any pyrimidines it wont cut..

But someone with more knowledge then me can for sure answer this.

I just wonderd why they call it an apurinic site since the pyrimidines are cut away and not the purines.

josse on Tue May 3 21:20:55 2011 said:

You're absolutely right, it removes damaged pyrimidines. I got confused exactly because of what you said (that it creates apurinic sites). So, then the question is: does it remove damaged purines as well?
I want to test a protocol used on a Neanderthal specimen where they tried to repair the DNA when preparing their next generation sequencing libraries. They used UDG and Endo VIII which sounds pretty extreme to me. Since Uracil is by far the most frequent damage, why not using UDG and then a PolI and a ligase?

Does anyone knows what PolI Klenow does when it meets an abasic site on single stranded DNA? Does it stop or does it incorportate a base randomly?