TI - NIR activity in cell -free extracts . AB - Reconstitution of DNA repair in whole cell extracts suggests that BER is the major pathway for removal of oxidative DNA base damage ( 41,42 ) . However , kinetic data indicate that both Ape1 and the DNA glycosylases/AP lyases can act directly on DHU*G . To understand which of the two pathways is responsible for repair of the majority of oxidative DNA base damage in vivo , we compared AP endonuclease and DNA glycosylase activities towards 3'-[32P]dCMP-labelled DHU*G in HeLa cell -free extracts under different reaction conditions . As shown in Figure 4A , at pH 7.6 , with or without 2 mM MgCl2 , we detect mainly a DNA glycosylase activity ( lanes 3 and 4 ) . Addition of 0.3 mM ZnCl2 increases the NIR activity up to the level of the DNA glycosylase -dependent incision ( lane 5 ) . However , at pH 6.8 , NIR was the major activity ( lane 6 ) . Similar results were obtained when 0.5 mM MgCl2 was substituted for 0.3 mM ZnCl2 . Interestingly , the level of BER activity remained constant under the various reaction conditions ( Fig 4A ) . To investigate whether the faster migrating fragments observed below the NIR reaction product in Figure 4A ( lanes 5 and 6 ) might be due to nuclease degradation from the 5' end of DHU*G , the HeLa cell -free extracts were incubated under various reaction conditions with the same 30mer oligonucleotide , but 5'-32P-end-labelled . Under these conditions a 10mer fragment was obtained ( Fig 4B , lanes 6-11 ) . As purified Nfo and Ape1 also generate 10mer products , it is likely that the SUBstrate oligonucleotide was cleaved by an AP endonuclease present in the crude extract ( Fig 4B , lanes 4 and 5 ) . This result excludes the possibility that the 3'-labelled NIR product generated by cell -free extracts is due to non-specific 5'-3' exonuclease degradation and supports our belief that the migration pattern of incision products is consistent with the NIR mode of incision with subsequent 5'-3' exonucleolytic degradation of the NIR fragment . These results also suggest that AP endonuclease and DNA glycosylase activities in cell -free extracts can vary depending on the reaction conditions showing that the respective activities are finely tuned .