AAG plays an important role in the maintenance of genomic integrity, presumably through its ability to recognize, bind, and excise a wide range of DNA base lesions. It was therefore surprising that AAG also has the ability to recognize and bind a number of DNA base lesions that it is incapable of excising, in particular the ϵC lesion. Moreover, the tight binding of AAG to ϵC leads to the inhibition of its catalytic activity and, in addition, is known to shield ϵC from ABH2-mediated direct reversal repair.6
To understand the structural basis for the inhibition of AAG by ϵC-containing DNA, we solved the crystal structure of Δ79AAG bound to a 13-mer ϵC:G (ϵC paired opposite G) duplex.
Given that AAG can bind ϵC-containing DNA, we anticipated that the lack of activity may be a result of one or more of the following factors. (i) AAG might fail to flip ϵC into its active site, or it might flip ϵC into an alternative binding pocket that lacks the appropriate catalytic residues; (ii) the binding mode of ϵC in the active site might not favor accommodation of the water molecule thought to act as a nucleophile in the reaction; (iii) the side chain of the putative catalytic base (Glu-125) might adopt a non-productive conformation that fails to activate the putative catalytic water molecule; (iv) AAG might be unable to protonate ϵC, failing to activate it for departure.
The crystal structure shows that AAG successfully flips the ϵC inhibitor into the same active site pocket that binds the ϵA substrate, ruling out the first possibility. We also find that the putative catalytic water molecule is present in the inhibitor complex, ruling out the second possibility. Furthermore, as was observed in the structure of the Δ79AAG-ϵA:T substrate complex, this water molecule is in contact with Glu-125, as would be required for its activation. The water molecules are ideally positioned to attack the N-glycosidic bond. Thus, we infer that the inability of AAG to remove ϵC is unlikely to be due to a problem with nucleophilic activation or attack, ruling out the third possibility from our list.
We next examined the fourth possibility, that the failure to excise ϵC is due to a problem with leaving group activation. In a previous biochemical study, O'Brien and Ellenberger (11
) measured the pH rate profiles for the excision of neutral Hx and ϵA lesions by AAG and for its excision of the positively charged 7-meG lesion under single turnover conditions. They found that the pH rate profiles for ϵA and Hx excision follow a bell-shaped curve, indicating that for the excision of neutral lesions, AAG uses the action of both a general acid and a general base (Glu-125). The general base can activate a catalytic water molecule, whereas the general acid is expected to facilitate the protonation of neutral lesions, making the lesion base a better leaving group (11
). In contrast, the pH rate profile for the excision of 7-meG shows only a single ionization corresponding to a general base, suggesting that leaving group activation of 7-meG is not necessary because the base is already positively charged. To help pinpoint the site of protonation, the activity of AAG on Hx was compared with its activity on 7-deaza-Hx, and although AAG greatly enhances the rate of Hx excision (~108
), the same lesion with N7 changed to C7 is not cleaved by AAG, directly implicating the involvement of the N7 position in catalysis (see B
for numbering) (11
). Although this study was unable to identify a specific residue as the general acid, the crystal structure of a Δ79AAG(E125Q)-ϵA:T substrate complex shows a water molecule in contact with the equivalent position to N7 of Hx, that is, N7 of ϵA (A
), raising the possibility that a protein-bound water molecule could be responsible for protonation. Once protonated, the AAG active site is designed to stabilize the protonated form of the base through a hydrogen bond between N7H of ϵA and the backbone carbonyl oxygen of Ala-134 (A
). Given these findings on the catalytic significance of protonation of the N7 of Hx and ϵA, it is important to consider the equivalent position in the ϵC base. A superposition shows that unlike ϵA, ϵC has a carbon (C5) in the position equivalent to N7 and thus cannot be protonated at that site (A
). Therefore, as opposed to our findings with respect to possibilities one through three, it appears that the failure of AAG to cleave ϵC could be due to an inability to activate the ϵC leaving group by protonation. Because AAG is reported to bind and not cleave a number of different pyrimidine lesions, including 3-methyluracil, 3-ethyluracil, and 3-methylthymine (15
), this mechanism of inhibition may be broadly applicable.
FIGURE 4. Activation of the leaving group by protonation. A, superposition of the structure of the Δ79AAG-ϵC:G inhibitor complex (amino acid carbons in green; DNA carbons in yellow; and water as a red sphere) with that of the Δ79AAG-E125Q-ϵA:T (more ...)
Given that AAG cannot repair ϵC lesions, it is interesting that AAG binds this lesion so tightly. The molecular basis for the approximate 2-fold higher affinity of AAG for the ϵC:G duplex (as compared with the substrate ϵA:T duplex) can be attributed to an additional hydrogen bond formed between the carboxamide side chain of Asn-169 and the O2
of ϵC. Mutation of Asn-169 to residues that cannot maintain this hydrogen bond (Leu and Ala) completely abolished this 2-fold binding effect (), suggesting that this one hydrogen bond is chiefly responsible for the higher affinity of ϵC-containing DNA. Thus, in addition to its previously proposed role of serving to help discriminate between damaged and undamaged guanine (24
), Asn-169 appears to play a role in the recognition and binding of pyrimidine DNA lesions. It is important to note that Asn-169 is strictly conserved among AAG-related glycosylases (9
Although inhibition of AAG by divalent metal ions (Mg2+
, and Ni2+
) has been well documented (12
), no previous crystal structure displayed electron density consistent with such an ion, although these cations were used in the crystallization buffers (9
). Here we find density consistent with the presence of Mn2+
in position to coordinate the base opposite the ϵC lesion (G19) (C
and supplemental Fig. S4
). Binding of Mn2+
to this site appears to influence the pucker of the sugar, yielding a C2′-exo configuration (supplemental Fig. S4B
). This occurrence is the first time that this sugar pucker has been observed in an AAG structure. In our Mn2+
-containing structure, the binding of ϵC to the active site is nearly identical to that observed in the Δ79AAG-ϵA:T structure. Thus, the inhibitory effect of Mn2+
does not appear to be due to a large conformational change in the active site, although the electrostatics of the active site could be affected by the presence of the positively charged ion ~16 Å away. Other dynamic movements of protein or DNA that are important in catalysis might also be affected by the Mn2+
coordination. Although binding of divalent metal cations to protein and DNA is common, it is intriguing that we find this divalent metal bound to such an important site in this protein-DNA complex. We now have a physical model for the influence of divalent cations on AAG activity that can be tested.
With a molecular view of the AAG-ϵC abortive complex in hand, it is interesting to consider what the physiological benefits in forming this complex might be. Abortive complexes between alkyltransferase-like (ATL) proteins and a lesion that it cannot excise, O6
-alkylguanine, have recently been observed (25
). ATLs are known to interact with proteins in another DNA repair pathway, nucleotide excision repair, suggesting that ATLs may function to present alkylated DNA to nucleotide excision repair proteins for repair (25
). Given the difficulty of finding a single damaged DNA base in the midst of the genome, it makes sense that once found, some DNA repair proteins may be designed to “hand over” lesion bases that they cannot themselves repair to an alternative repair pathway. Although it is too early to infer directly from ATLs, preliminary data do show that AAG can interact with human nucleotide excision repair proteins hHR23A and hHR23B (27
). Obviously, more studies are necessary to establish whether AAG-ϵC abortive complexes interact with nucleotide excision repair proteins, resulting in ϵC repair, but this idea is intriguing and, given the recent studies on ATL, not without precedence. Originally identified by its ability to excise alkylated 3-methyladenine and 7-meG lesions (8
), the role of AAG in DNA repair is far more complex than once thought. Given the importance of repairing reactive oxygen and nitrogen species-generated DNA damage for tissues undergoing chronic inflammation, a complete understanding of the physiological function of AAG is essential.