TI - Substrate specificity of Ape1 . AB - Since our previous study failed to detect Ape1 -catalysed NIR ( 22 ) , we analysed the SUBstrate specificity of rec-Ape1 under the reaction conditions used for the purified protein from HeLa cells (HeLa-Ape1) . As shown in Figure 2 , rec-Ape1 , along with Nfo and HeLa-Ape1 , cleaved 5ohU*G , DHU*G , THF*T , alphaA*T , alphaT*A and DHT*A oligonucleotide duplexes ( Fig 2A and B , lanes 4 and 9 and lanes 3 , 7 and 11 , respectively , and data not shown ) . In addition , rec-Ape1 incises damaged supercoiled plasmid DNA containing either methylformamidopyrimidines or oxidized bases ( Supplementary Material , Fig 2 ) . We did not observe any difference in the activity of the Ape1 proteins when either 0.3 mM ZnCl2 or 0.5 mM MgCl2 was used , in agreement with published data ( 39 ) . In order to determine the mechanism of Ape1 -catalysed incision , the DNA fragments generated by the E.coli proteins Nth and Nfo were used as size markers . As shown in Figure 2A , Nth ( a DNA glycosylase/AP lyase ) generates 15 or 20mer fragments from 3'-labelled 5ohU*G and DHU*G oligonucleotides , respectively ( Fig 2A , lanes 2 and 7 ) . In contrast , Nfo ( an AP endonuclease ) generates 16 and 21mer fragments containing the dangling base , from the same SUBstrates ( Fig 2A , lanes 3 and 8 ) . Thus , the migration pattern of Ape1-cleavage products corresponds to that of Nfo , indicating a DNA glycosylase -independent , endonucleolytic mechanism of action ( Fig 2A and B , lanes 4 , 5 , 9 and 10 and lanes 3 , 4 , 7 , 8 , 11 and 12 , respectively ) . Taken together these results confirm that the NIR activity is indeed due to Ape1 .