NTH1 and OGG1, which repair oxidized bases in mammalian cells, belong to the Nth family of DNA glycosylases, and carry out β elimination-mediated DNA strand cleavage to produce 3′-phospho α,β-unsaturated aldehyde (3′ PUA) terminus at the site of the damaged base. In contrast, the more recently characterized mammalian DNA glycosylases NEIL1 and NEIL2, which belong to the Nei family, produce 3′ phosphate containing terminus after βδ elimination reaction at the AP site. Both types of termini (3′ PUA and 3′ P) are efficiently removed by E. coli
APEs to produce the 3′ OH necessary for DNA repair synthesis. APE1, the only APE in mammalian cells, was believed to play a central role in all types of BER processes, like E. coli
]. However, we showed recently that APE1 is dispensable when BER is initiated by NEIL1 [12
]. Here we show that APE1 is similarly dispensable in NEIL2-initiated repair.
In this study we demonstrate that NEIL2 behaves much like NEIL1 in directly interacting with the other BER components, specifically Pol β, Lig IIIα and XRCC1. Because NEIL1 and NEIL2 share only limited sequence homology, it is remarkable that the same regions of Pol β and Lig IIIα are involved in interaction with both NEILs. Far-Western analyses revealed that the interaction region in Lig IIIα lies within the C-terminal 175 amino acids, and the interaction region in Pol β is located within the N-terminal 140 amino acid residues. The physiological significance of both NEILs' binding to the same region of Pol β and Lig IIIα warrants further investigation. This is the first report showing direct physical association of human NEIL2 with Lig IIIα and Pol β. A recent report of mouse NEIL2 and Pol β's binding to microtubules further supports their close association [33
]. However NEIL2 failed to show direct interaction with PNK (), although PNK was stably associated with NEIL2 in a larger complex including the other BER components. Furthermore, XRCC1 alone interacts weakly with the NTD of NEIL2, but the interaction is significantly enhanced when the other BER proteins are present. Association of NEIL2 with XRCC1 has also been recently reported [34
An X-ray crystallographic structure of an enzymatically active deletion mutant of NEIL1, lacking 56 C-terminal residues suggests that its C-terminal region is unstructured [35
]. We have shown earlier that the interacting region of NEIL1 lies within its C-terminal domain (between residues 289 and 350) [12
]. Thus NEIL1'
s interacting domain for Polβ and Lig IIIα may have a flexible structure. The tertiary structure of NEIL2 has not yet been elucidated. However, limited proteolytic digestion suggests that there are two major domains in NEIL2, the NTD (spanning residues 1–198) and the CTD (residues 199–331) (). Our data further reveal that in case of NEIL2, the 133 C-terminal residues, including the zinc finger motif that is necessary for DNA binding and catalysis [36
], is dispensable for interaction with Polβ, Lig IIIα () and XRCC1 polypeptides (). It is intriguing that the interacting domain of NEIL1 for the same proteins is localized in its C-terminal domain, which is dispensable for its in vitro
glycosylase/lyase activity [12
]. In contrast, not only is the C-terminal domain of NEIL2 essential, but the interacting domain, which is at the N-terminus, is also indispensable for activity.
Our in vivo
data () further showed that NEIL2 associates with the other BER proteins, Polβ, Lig IIIα and PNK as well as XRCC1, but not with APE1. We reported previously that NEIL1-mediated repair is dependent on PNK, but not on APE1, while NTH1-initiated repair is APE1-dependent [12
]. In the present study we were able to reconstitute NEIL2-initiated repair, and established this repair to be PNK-dependent. XRCC1 was not required for these in vitro
BER reactions and thus was not used in the reconstitution studies; however it is most probably required for assembly of the NEIL2 repair complexes in cells [37
Finally we made the significant observation that the FLAG-NEIL2 immuno-complex was capable of completing repair of 5-OHU-containing plasmid DNA (). This multi-protein complex has to be dynamic, because the same NEIL2 molecule cannot simultaneously interact with multiple proteins using its common interacting domain. A possible explanation for such a complex is that it allows coordinated hand-off among the repair proteins. However, the stoichiometry of the component proteins in this complex needs to be determined. Taken together, the association of NEIL2 with downstream BER proteins, Pol β, Lig IIIα and PNK, like NEIL1, supports a model of repair coordination in which NEIL2 recruits these proteins, and forms a larger repair complex at the site of DNA damage.
The NEILs are thus functionally distinct from the previously characterized oxidized base specific DNA glycosylases, NTH1 and OGG1. Distinctive feature of NEILs versus NTH1/OGG1 are that NEILs prefer excising lesions from DNA bubble structures, whereas both NTH1 and OGG1 are active only on duplex DNA [38
]. The dichotomy in the preference of NEILs and NTH1/OGG1 for bubble versus duplex DNA raises the possibility that NEILs have preferred function in repairing base lesions present in transient transcription or replication bubbles. The NEIL1-deficient mouse embryonic fibroblasts were found to be sensitive to radiation [39
], providing support for NEIL initiated BER in preventing the genotoxicity of oxidative stress. Recently Stephen Lloyd's group generated NEIL1 knockout mice, which exhibited high levels of mitochondrial DNA damage. These knockout animals also developed severe metabolic syndrome [40
]. It would be interesting to investigate the organismal consequences associated with deficiency in NEIL2. The biological relevance of the NEIL/PNK-dependant repair pathway was further supported by results showing that PNK-deficient A549 cells display an elevated mutation frequency and are sensitive to ionizing radiation [41
]. Thus, PNK-dependent repair of NEILs may play a unique role in maintaining the functional integrity of mammalian genomes.