TI - DISCUSSION . AB - Oxygen radicals generated as by-products of cellular metabolism cause extensive damage to DNA . Oxidative DNA damage have been postulated to be the major type of endogenous damage leading to human degenerative disorders including cancer , cardiovascular disease and brain dysfunction . Following the acquisition of the nucleus during the formation of the eukaryotic cell , oxidative metabolism was confined to the mitochondria , thus creating pseudo-anoxic conditions and protecting the genome located in the nucleus ( 43 ) . Since the eukaryotic nucleus is a poorly oxygenated cellular compartment ( 44 ) , oxidative DNA damage preferentially formed under anoxic conditions might result in significant biological consequences in vivo . Interestingly , cancer cells can grow under reduced oxygen-supply conditions and tumour hypoxia is associated with poor prognosis and resistance to radiation therapy ( 45 ) . In contrast to oxidatively damaged bases such as 8-oxoguanine and thymine glycol , formation of 5 , 6-dihydropyrimidines and alpha-anomeric 2'-deoxynucleotides requires strict anoxic condition ( 1,2 ) . This might suggest that the latter lesions are biologically important DNA damage formed in the eukaryotic nucleus in vivo . In NIR , an Nfo/Apn1-like damage-specific endonuclease directly nicks DNA containing free radical -induced base lesions acting as a back-up repair pathway to BER ( 22 ) . As the identity of the human functional homologue of Nfo was unknown , we proceeded to purify the enzyme responsible for the incision of DHU*G oligonucleotide duplexes from HeLa cell extracts . Following seven chromatographic steps , we identified this activity as belonging to Ape1 ( Fig 1 ) . To characterize the SUBstrate specificity of Ape1 , we compared the activity of HeLa-purified and recombinant proteins towards various free radical -induced base lesions . The DNA duplexes containing DHT*A , DHU*G , 5ohU*G , THF*T , alphaA*T and alphaT*A were readily incised by both Ape1 proteins . Like Nfo and Apn1 , Ape1 nicks DNA on the 5' side of the damaged base , leaving the dangling damaged nucleotide on the 5'-terminus of the downstream reaction product ( Fig 2 ) . Taken together the results show that Ape1 is involved in the NIR pathway for oxidative DNA base damage . In contrast to both E.coli and humans , the major AP endonuclease of the budding yeast is Apn1 , an Nfo homologue ( 46 ) . While the reason for the reversal in AP endonuclease prevalence is not understood , here we provide evidence that Ape1 is in fact the mammalian functional counterpart of Apn1 , rather than E.coli Xth . Thus , in eukaryotes the major AP endonuclease is also involved in the DNA glycosylase -independent repair pathway . Dramatic differences in reaction conditions were found for AP endonuclease and NIR activities of Ape1 ( Fig 3 ) . Thus , the AP endonuclease activity was maximal between pH 7.8 and 8.2 , while NIR activity was maximal between pH 6.4 and 6.8 ( Fig 3B ) , conditions that are also optimal for AP site-DNA binding ( 47 ) . Likewise , AP endonuclease activity was maximal over a broad range of KCl concentrations ( 25-200 mM ) , whereas NIR activity decreased dramatically when the KCl concentration exceeded 50 mM (Fig 3C) . Interestingly , optimal conditions for NIR activity are similar to those for the 3'-5' exonuclease activity of Ape1 observed on matched , 3'-mispaired and nucleoside analogue beta - l-dioxolane-cytidine terminated nicked DNA ( 30 ) . In agreement with previous data for AP endonuclease activity of Ape1 ( 25,40 ) over the concentration range 1-5 mM , Ape1 -mediated cleavage of THF*G and DHU*G shows a sigmoid dependency on MgCl2 (Fig 3A) . Furthermore , MgCl2 titration led to an increase in conformation related fluorescence of Ape1 but not of Apn1 (Fig 3C) . Based on this it is tempting to speculate that Ape1 could be an allosteric enzyme , regulated by the Mg2+-effector . Although , initial structural studies indicated that the active site of Ape1 is unlikely to undergo radical structural changes upon Mg2+ binding ( 48,49 ) . A more recent study , on the X-ray structure of the full-length Ape1 protein crystals made at different pH reveals two metal ions bound 5 apart in the active site at pH 7.5 ( 47 ) , whereas only one metal ion is bound at acidic pH . In addition , the loop regions of Ape1 , consisting of residues 100-110 and 120-125 , exhibit significant structural variation ( 47 ) . Therefore , we hypothesize that Mg2+ -dependent conformational change regulates catalytic activity of Ape1 and may channel the repair of oxidative DNA damage to either the BER or NIR pathways . However , structural studies of the NIR complex are required to fully investigate these phenomena . Analysis of the kinetic data presented in Table 1 indicates that the primary SUBstrate for Ape1 is a THF residue and although reduced under the NIR-conditions , the AP endonuclease activity of Ape1 is still robust . The kcat/KM value of Ape1 acting upon DHU is 1.5-fold more efficient than the value for hNth1 (50) , indicating that DHU might be processed equally well by both BER and NIR ( Table 1 ) . Overall , a comparison of the kinetic parameters for the BER and NIR activities demonstrates that Ape1 can efficiently back-up the DNA glycosylases to repair DHU adducts . To determine the balance between BER and NIR pathways , we measured activities towards DHU*G in HeLa cell -free extracts under different reaction conditions . We found that , at pH 6.8 in the presence of 0.3 mM ZnCl2 , NIR was the major activity in the extracts ( Fig 4A , lane 6 ) . We propose that under certain conditions the extremely high cellular concentration of Ape1 ( 35 x 105-7 x 106 molecules/cell ) ( 51 ) may direct the processing of oxidative DNA damage to the NIR pathway in vivo . A 5'-3' exonucleolytic degradation of the NIR fragment can be seen in cell -free extracts ( Fig 4A , lanes 5 and 6 ) . This activity might be due to FEN-1 . Indeed , FEN-1 exonuclease activity is stimulated at low Mg2+ and monovalent salt concentrations ( 52 ) . However , to exclude the possibility that the NIR fragment emerges from non-specific 5'-3' exonuclease activity , we used a 5'-labelled DHU*G duplex oligonucleotide in our assay . As expected , incubation of the 30mer oligonucleotide in HeLa cell -free extracts under different reaction conditions gave rise to a 10mer fragment retaining 5'-label ( Fig 4B , lanes 6 and 8-11 ) . These results exclude the possibility that the NIR fragment generated by the purified Ape1 proteins and cell -free extracts is due to a 5'-3' non-specific exonuclease halting at the modified site . Taken together the results suggest that the repair mode for oxidative DNA damage in vivo depends upon the intracellular environment and concentration of a given repair enzyme . As the first 61 N-terminal residues of Ape1 are indispensable for redox but not for AP endonuclease activity ( 38,53 ) , we generated a truncated Ape1 lacking the N-terminal 61 amino acids (NDelta61-Ape1) and assayed it for AP endonuclease and NIR activities . Although NDelta61-Ape1 exhibited no decrease in THF*G incision , we observed a 2.5 - and 20-fold decrease in NIR activity towards DHU*G and alphaA*T , respectively , compared with wild type Ape1 ( Fig 5 ) ( 38 ) . The results indicate that the redox domain of Ape1 regulates the NIR activity , thus providing insight for an additional role of the Ref domain . Importantly , in known Ape1 crystals the N-terminal 35-42 residues were truncated or not visible in the electronic density maps and the final models of Ape1 ( 47,48 ) . In conclusion , it is tempting to speculate that the Ref domain was acquired during evolution by an AP endonuclease to repair oxidative damage to both DNA bases and proteins . Evolutionary conservation of two types of AP endonucleases with overlapping SUBstrate specificity [Xth and Nfo in Ecoli , Apn1 and Apn2 in yeast and Ape1 and Ape2 in human cells ( 29 ) ] , highlights the existence of two alternative repair pathways for oxidative DNA damage . However , the biological impact of the different Ape1-DNA repair activities , including NIR , needs further investigation . As Ape1-initiated NIR can excise alpha-anomeric nucleotides , DNA adducts that are not SUBstrates for BER , we suggest that NIR targets oxidative DNA damage formed under natural ( anoxic ) conditions . Therefore , as NIR and BER also share many common SUBstrates , we propose that they work in concert to cleanse genomic DNA of potentially mutagenic and cytotoxic lesions . This in part , may explain the lack of readily discernable phenotype of DNA glycosylase-deficient mice ( 17 ) and the increased susceptibility to oxidative stress of Ape1 heterozygous null mutant mice ( 54 ) . Figure 2 Comparison of SUBstrate specificities of rec-Ape1 and HeLa - purified Ape1 . Reactions were performed under standard NIR condition. ( A ) [3'-32P] dCMP-labelled 5ohU*G and DHU*G ; ( B ) 5'-32P-labelled THF*T , alphaA*T and alphaT*A . Figure 3 Activity profiles and conformational changes of Ape1. (A-D) Ape1 nucleotide incision ( squares , straight line ) and AP-endonuclease ( triangles , dotted line ) activities depending on Mg2+ concentrations ( A ) , pH profile ( B ) and ionic strength ( C ) . ( D ) Mg2+ -induced changes in the intrinsic tryptophan-fluorescence of Ape1 ( filled circle ) and Apn1 ( empty circle ) . Figure 4 DNA repair assay using whole whole cell extracts from HeLa cells . The extracts were incubated with 3' - ( A ) or 5'-labelled ( B ) DHU*G SUBstrates under standard NIR or AP-endonuclease conditions with different concentrations of metal ions . Figure 5 DNA repair assay of the N61Delta-Ape1 mutant and wild-type Ape1 proteins . Reactions were performed under standard AP-endonuclease ( lanes 1-3 ) or NIR ( lanes 4-12 ) conditions using 3'-labelled oligonucleotide SUBstrates . In the case of THF*G duplex concentrations of Ape1 and N61Delta-Ape1 were 5 ( lanes 2 and 3 ) andP 50 pM ( lanes 4 and 5 ) . Table 1 .