The broad view of BER in the last decade was that of a sequential process in which individual repair enzymes carried out reactions independently of one another [112
]. Based on the emerging evidence for the existence of the ‘protein interactome’, we propose a new paradigm in BER that postulates collaboration of multiple proteins in coordinated fashion involving specific protein-protein interactions to enhance the efficiency of repair. It is now evident that cellular processes involving repair of both endogenous and induced genomic damage are essential for maintaining genomic integrity and homeostasis, which involve dynamic and complex interactions among a multitude of proteins in the cellular ‘proteome’ in response to both endogenous cellular activities and environmental stress [160
]. Such protein complexes are not unique to BER, and similar situations appear to exist for other DNA excision repair pathways. There is increasing evidence that the mammalian BER proteins are organized in temporally controlled complexes for cross-talks among themselves and with DNA, presumably for optimum efficiency of damaged base recognition and repair.
Formation of the multiprotein complexes for oxidative damage repair is enhanced by ROS [51
]. It was previously shown that NEILs carry out SN-BER mediated by PNK, Polβ, DNA Lig IIIα and XRCC1 by stably interacting with Polβ, DNA Lig IIIα and XRCC1 [51
]. Other interactions between OGG1 and XRCC1 was reported earlier [162
]. It now appears that additional proteins including those of the DNA replication machinery are also involved in BER [26
]. Several DNA glycosylases interact with replication related-proteins, namely, PCNA and replication protein A (RPA). MYH and UNG interact with both PCNA and RPA [164
]. We observed stable and functional association between NEIL1 and WRN protein [167
]. Although these associations may be merely for carrying out LP-BER, several observations, e.g., (i) colocalization of UNG-PCNA-RPA complex at replication foci and (ii) S-phase-specific increase in the levels of UNG2, MYH and NEIL1 [50
], supports our hypothesis that certain glycosylases are involved in RA-BER.
A key unanswered question is whether there is coordinated hand-over among interacting partners in smaller sub-complexes or a larger complex is preformed to carry out efficient repair. The coordination in protein complex formation was originally compared to the passing of the baton, where the repair product of each enzyme in the BER pathway is “handed” over to the next enzyme in the pathway [169
]. It would be important to unravel how NEIL1 or any DNA glycosylase can simultaneously and binarily interact with several downstream proteins in the repair pathway initiated by NEIL1. All of NEIL1’s interacting partners identified so far bind to the C-terminus of NEIL1 [51
]. Though NEIL1’s interaction with its multiprotein partners using a common interaction interface (C-terminus) may be explained based the unfolded conformation/ flexibility of its C-terminus, further structural characterization of the C-terminus of NEIL1 and structure-function studies are required to elucidate the mechanism in detail.
Unique Structural features of mammalian DNA glycosylases
Mammalian DNA glycosylases possess unique structural features compared their counterparts in lower organisms and bacteria. The major characteristic of mammalian glycosylases (which may also be true for other mammalian BER enzymes) is an unfolded extension or tail which participates in subcellular translocation, and more importantly in protein-protein interactions. The sequence alignment of hNTH1 with NTHs from E. coli,
archea and other lower organisms shows that the human and other mammalian NTH1 has an N-terminal tail, absent in E. coli
and archea. This N-terminal tail (residues 1–95) contains putative nuclear and mitochondrial localization signals [170
]. hNTH1 was found to have low activity compared to E. coli
]. This is because of the inhibitory role of N-terminal tail of hNTH1 on its turnover. Thus, the N-terminal tail of hNTH1 could regulate the overall catalytic turnover by reducing the rate of product release without affecting glycosylase or AP lyase activities [172
The crystal structure of an enzymatically active deletion construct of hNEIL1 (lacking 56 C-terminal residues) and other modeling studies indicated that hNEIL1 has an unfolded C-terminal extension (~100 residues) which is absent in bacteria Nei [173
]. The crystal structure of hNEIL1 also indicates the presence of internal Zn-less finger, unlike the C-terminal Zn finger present in other Nei orthologs. Our data show that NEIL1 interacts with downstream SN-BER proteins, via its unfolded C-terminal extension (residues 289-349) [115
We used the software of Molecular Kinetics, Inc. [174
] for prediction of prediction of naturally disordered regions (PONDR) to compare the secondary structures of hNEIL1 and hNTH1 with their E. coli
prototypes Nei and Nth respectively (). Extended disordered regions are present only in the human proteins and their location at the N-terminus of hNTH1 and at C-terminus of hNEIL1 that are consistent with the experimental evidence about the absence of defined structure using X-ray crystallography. The prediction about the disordered structure in the C-terminal region of NEIL1 supports the hypothesis that the region individually interacting with multiple proteins is intrinsically disordered.
Figure 3 PONDR Plot of the predicted secondary structures of hNEIL1, hNTH1 and their E. coli prototypes, endonuclease VIII (Nei) and endonuclease III (Nth) respectively. The protein sequences were obtained from NCBI database. PONDR. score of 0.5 and higher indicates (more ...)
shows the PONDR©plot of the predicted secondary structure in hNEIL1 and hNTH1. The disordered structures of hNEIL1 at the C-terminus and of hNTH1 at the N-terminus are absent in their E. coli prototypes.
DNA glycosylases interact with non-BER proteins
Several other non-BER proteins have also been shown to be involved in BER, however their precise in vivo
role in repair is yet to be understood. NEIL2 interacts with YB-1, a Y-box binding protein and it was suggested that YB-1 may be required for the fine-tuning of repair [161
]. NTH1 was also shown to interact with and stimulated by YB-1 [175
]. We have recently shown a stable interaction between NEIL1 and 9-1-1 complex and NEIL1’s stimulation as a result of this interaction [75
]. 9-1-1 is a heterotrimeric protein complex and it acts as a sliding clamp similar to PCNA, but is activated by DNA damage and cell cycle checkpoints. NEIL1’s interaction with 9-1-1 highlights the linkage between NEIL1-initiated BER and damage signaling pathways. Our recent studies also showed that NEIL1’s stable association with PCNA [74
]. It is highly likely that NEIL1’s interactome contains a large number of non BER proteins. Whether and how these proteins participate in BER poses an important topic for future investigation.