Studies on DNA containing randomly generated lesions established that FapyG and FapyA are substrates for various OG glycosylases (34
). However, due to limitations in studying randomly generated lesions, the absolute efficiency of repair of these lesions was unknown. Moreover, the magnitude of the effects of the base opposite the lesions could not be established. Using defined oligonucleotide duplex substrates containing formamidopyrimidine lesions and quantitative kinetics, such features have now been revealed. An important aspect of the analysis of the activity of the glycosylases in this study was the evaluation of the glycosylase reaction separately from the strand scission (lyase) reaction. With Fpg these two reactions are tightly coupled; however, with hOGG1 and Ntg1 the glycosylase step is much faster than the lyase step. In fact, the extent of the activity of these enzymes may be underestimated or differences in substrate preferences may not be revealed if the enzyme is used to provide strand scission. This can be illustrated with hOGG1 in which the relative preference for C over A opposite the lesion is larger when comparing the rate constants for the glycosylase reaction (kg
) than the lyase reaction (kgl
). Similarly, the preference of Ntg1 for removal of FapyG in the promutagenic FapyG:A bp over the FapyG:C bp is only revealed when analyzing the glycosylase reaction.
Importantly, this approach revealed the robust activity of Fpg and hOGG1 for removal of FapyG lesions. Indeed, the measured rate constants show that Fpg excises FapyG lesions much more efficiently than OG. Similarly, FapyG was removed as efficiently as OG by the human BER glycosylase hOGG1. The fact that both lesions are substrates for Fpg and hOGG1 may be rationalized by the structural similarity between FapyG and OG. Indeed, though the structures of hOGG1 and Fpg are completely different from each other, the strategy for OG recognition by the two enzymes is similar (9
). Both enzymes recognize OG by extracting the lesion base from the helix for placement within a lesion specific pocket where contacts are made to NH7 of OG which distinguishes it from G (48
). X-ray structural studies of Lactococcus lactis
Fpg (LlFpg) bound to lesion-containing DNA have shown that a carbocyclic analogue of FapyG is specifically recognized within the OG base-binding site by residues that are strictly conserved among Fpg enzymes from different species (48
). However, the manner of recognition of FapyG within the LlFpg active site was found to be significantly different than that observed for OG recognition by Bacillus stearothermophilus
Fpg (BsFpg). The flexibility to adopt alternate recognition complexes may be a feature that enables Fpg to recognize a wide variety of substrates (26
). The ability of Fpg to remove FapyA and FapyG with similar efficiencies also is consistent with the idea that the Fpg base recognition site is open and plastic. In contrast, X-ray structural studies of hOGG1 bound to DNA substrates have revealed a rigid, preformed site into which OG fits in snugly (9
). There are many contacts with the Watson–Crick face of OG, as well as residues that interact on opposite sides of the planar OG heterocycle to sandwich the base within the active site. Calculations have also indicated the importance of favorable dipole–dipole interactions in hOGG1 recognition of OG (51
). The minimal activity of hOGG1 toward removal of FapyA lesions is consistent with a restricted substrate scope that is limited to lesions that are very similar to OG. The “A”-like features of FapyA may not allow for proper engagement in the OG-specific pocket of hOGG1 to allow for efficient excision (9
). The fact that FapyA is only removed when paired with C is also consistent with the preference of hOGG1 for lesions derived from G that would be found paired with C.
The identity of the base opposite has a marked influence of the observed rate of lesion excision by Fpg and hOGG1 (). Single-turnover experiments revealed that both enzymes more readily remove FapyG or OG when paired with C over A. Similar results were previously observed in the steady-state kinetics studies with Fpg with FapyG containing substrates (43
). This selectivity for FapyG and OG in the correct Watson–Crick context provides a means of protecting the genome from mutagenesis. Selective removal of FapyG or OG opposite C helps in restoring the original base pair, while slower removal of the lesions opposite A prevents G →T transversion mutations. In the structural studies, hOGG1 and Fpg provide specific contacts to the intrahelical C left behind after expulsion of OG (49
). However, the extent of C-specific interactions is more extensive with hOGG1. This is consistent with a more stringent selection against the presence of A opposite OG by hOGG1. This is illustrated by the relative rate constants () that demonstrate a preference of hOGG1 for C over A for OG removal that is quite large (~3000-fold). Notably, the relative preference of hOGG1 for C over A opposite FapyG is significantly reduced (47-fold). This is due primarily to the greater ability of hOGG1 to remove FapyG from FapyG:A base pairs than OG from OG:A base pairs. The relative preference of Fpg for C over A when the G lesion is FapyG is also reduced compared to OG; however the magnitude of the difference is not quite as large as was observed with hOGG1.
Though often thought of as an OG glycosylase, Ntg1 shows minimal excision activity with OG (46
). However, Ntg1 catalyzes removal of both formamidopyrimidine lesions. In fact the observed rates show that the activity toward the FapyA lesion is quite efficient. The activity of Ntg1 toward FapyG is approximately 100-fold slower than FapyA; however the rate constants measured are similar to those of other glycosylases with standard substrates (46
). Ntg1 is similar in terms of sequence and substrate specificity to the E. coli
BER glycosylase endonuclease III and its human homologue, NTH1 (26
). Endonuclease III has been previously shown to be capable of removal of FapyA and FapyG, with more activity toward FapyA (54
). The fact that Ntg1 cleaves FapyG and FapyA is consistent with these lesions resembling pyrimidines. Though no structural data on Ntg1 is available, it is possible that the lack of activity toward OG is due to the inability of a large planar purine heterocycle to fit into the lesion-binding site. Another unusual feature of the glycosylase activity of Fpg and Ntg1 toward FapyA is insensitivity to the identity of the opposite base. For example, FapyA is removed as efficiently when paired with A, G or C as it is when base paired with T. Removal of a lesion from a promutagenic base pair leaves the incorrect nucleotide thus leading to permanent mutations. The fact that FapyA is weakly mutagenic may suggest that repair of this lesion may not be as crucial, and therefore highly discriminatory recognition of this lesion may not have been developed. Indeed, the activity of Fpg and Ntg1 toward removal of FapyA may be a consequence of the structural similarity of this lesion to other substrates (e.g., OG for Fpg and thymine glycol for Ntg1).
A factor that influences the recognition of a lesion and the opposite base is the stability of the lesion-containing base pair. If the lesion base pair is more easily disrupted, this may compensate for the absence of favorable interactions with the proper opposite base. Reduced selectivity for the base opposite a lesion has previously been observed in studies on Fpg excision of the helix-destabilizing hydantoin lesions, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) (45
). Duplexes containing the FapyG lesion do not appear to be less stable than those containing OG (20
), however, the local stability may be reduced due to the structural flexibility of the ring-opened FapyG relative to the planar OG. This property may also result in reduced stability at the damaged base pair upon interrogation by the glycosylase enzyme. Recent structural studies of BsFpg trapped via disulfide cross-linking to normal nonlesion DNA suggest that this glycosylase actively tests the robustness of base pairs as part of the damage search process (51
). Notably, FapyA lesions are more duplex destabilizing than FapyG (23
), and this may be a factor resulting in the lack of opposite base sensitivity with both Fpg and Ntg1.
An important aspect of the prevention of mutations associated with OG is the removal of misincorporated adenines from OG:A base pairs by the adenine glycosylase MutY. This enzyme intercepts this promutagenic base pair and allows for recreation of a proper substrate for Fpg. MutY has been shown to remove adenine from FapyG:A base pairs (43
). The rate of adenine removal mediated by MutY from FapyG:A bps is not as fast as from OG:A bps, but faster than when G is paired with A. Surprisingly, however, recent in vivo studies in E. coli
have shown that FapyG is not particularly mutagenic, and moreover this mutagenesis is not enhanced in mutM
– E. coli
. This suggests that the activity of MutY toward FapyG:A base pairs may not be as important for preventing mutagenesis as the activity toward OG:A. The origin of the low mutagenesis of FapyG was proposed to be due to the processing by the DNA polymerase. This proposal was based on detailed analysis of the kinetics of KF exo+
processing of FapyG-containing DNA templates (21
). The observed inefficient extension of Fapy-G:A base pairs coupled with the inherently lower insertion frequency would provide an opportunity for the exonuclease activity of the DNA polymerase to correct its mistakes. It is also possible that removal of FapyG may occur via alternative BER glycosylases or other repair pathways (e.g., NER). Redundant mechanisms for preventing mutations from a given lesion may make observing the effects of a specific repair enzyme difficult. Moreover, such redundancies are likely important to mitigate the properties of mutagenic and toxic lesions, like FapyG.
However, the striking observation that FapyG is more mutagenic in simian kidney (COS-7) cells than in E. coli
suggests that the processing of FapyG by polymerases and repair enzymes may be distinctly different in eukaryotic cells. Moreover, the presence of FapyG was also more mutagenic than OG in simian kidney cells. This is particularly interesting in light of the more relaxed specificity of hOGG1 for the proper opposite base when the lesion is FapyG. In fact, this may contribute to the higher mutation frequency of FapyG over OG lesions in COS-7 cells. Analysis of the details of the activity of other human glycosylases toward removal of FapyG and the effects of the base opposite may be quite illuminating. In particular, the mammalian NEIL1 glycosylase has been shown to be able to remove Fapy lesions (37
). This glycosylase appears to remove mainly damaged pyrimidines (55
) and therefore one might find that this enzyme also has activity toward FapyG in promutagenic contexts. Thus, detailed analysis of the kinetics of FapyG with other BER glycosylases in defined base pairing and sequence contexts may provide insight into their role as potential modifiers of mutagenesis caused by FapyG lesions. Moreover, such studies would aid in the design of cellular experiments to evaluate how such effects translate to a cellular environment.