Duplex DNA labeled on the lesion containing strand was incubated with purified Mag and the resulting complex was visualized by gel shift analysis. Mag was tested KU-55933 for its ability to bind duplexes containing the following: an εA, a 1,2 d cisplatin adduct, a Hx, a G:T mismatch or the AP site analogue tetrahydrofuron , for simplicity we refer to the THF as an AP site. As evidenced by the shifted bands, Mag showed strong binding to the oligonucleotide duplexes containing an AP site and significant binding to the duplexes containing an εA lesion. We had previously shown that the human AAG enzyme binds DNA oligonucleotides containing cisplatin cross linked DNA base adducts, although not as strongly as it binds εA containing DNA. Here we show that Mag also binds duplex DNA containing the 1,2 d cisplatin adduct, but, as for the AAG enzyme, Mag,s binding to this lesion was weaker than that for εA and AP site containing DNA.
Surprisingly, Mag exhibited no apparent binding to the duplex containing Hx in the random sequence context, and as expected Mag showed no binding to the undamaged duplex or that containing a G:T mismatch. In summary, Mag exhibited strong binding to the duplexes containing εA or an AP site, weak binding Belinostat to the duplex with 1,2 d cisplatin adduct and no apparent binding to the duplexes with Hx, with a G:T mismatch or with no damage. We went on to test whether and how well Mag excises these base lesions in this sequence context, excluding the AP site. Mag displayed robust activity for εA excision, and to our surprise, Mag also displayed significant Hx excision, albeit not as robust as that for εA. Interestingly, although Mag could bind 1,2 d cisplatin adducts, it did not cleave either of the glycosyl bonds associated with this intrastrand DNA cross link.
Finally, Mag activity on the undamaged duplex and on the duplex containing a G:T mismatch was undetectable. 3.2. Mag glycosylase activity in the presence of competitors In order to further explore Mag,s ability to recognize different substrates, we monitored Mag activity in the presence of various competitors. Mag mediated εA excision activity was followed as a function of time, in the presence of various competitors. Duplex DNA substrate with εA in the random sequence context was incubated with Mag in the presence of 2000 nM cold competitor DNA containing either an εA, an AP site, a Hx, a 1,2 d cisplatin adduct, a G:T mismatch or no damage at all and Mag activity on the εA containing substrate was followed as a function of time.
As expected, Mag exhibits maximal activity in the absence of any competitor and the addition of undamaged duplex DNA only slightly inhibited Mag,s activity on the εA substrate. However, unlabeled competitor duplexes containing εA or an AP site strongly inhibited Mag activity, and that containing the 1,2 d cisplatin adduct showed moderate inhibition of activity. Competition by Hx and G:T mismatch containing duplexes were similar to that by undamaged DNA. Taken together, the relative ability of each lesion to inhibit Mag,s base excision activity on an εA substrate, paralleled their ability to bind the lesion containing duplexes in our initial binding experiments. However, it should be noted that the glycosylase activity as measured here, reflects a combination of both lesion binding and glycosyl bond cleavage.