Efficient recognition of substrates and substrate analogs by the adenine glycosylase MutY requires the C-terminal domain

Abstract

The Escherichia coli DNA repair enzyme MutY plays an important role in the prevention of DNA mutations by removing misincorporated adenine residues from 7, 8-dihydro-8-oxo-2'-deoxyguanosine:2'-deoxyadenosine (OG:A) mispairs. The N-terminal domain of MutY (Stop 225, Met1-Lys225) has a sequence and structure that is characteristic of a superfamily of base excision repair glycosylases; however, MutY and its homologs contain a unique C-terminal domain. Previous studies have shown that the C-terminal domain confers specificity for OG:A substrates over G:A substrates and exhibits homology to the d(OG)TPase MutT, suggesting a role in OG recognition. In order to provide additional information on the importance of the C-terminal domain in damage recognition, we have investigated the kinetic properties of a form lacking this domain (Stop 225) under multiple- and single-turnover conditions. In addition, the interaction of Stop 225 with a series of non-cleavable substrate and product analogs was evaluated using gel retardation assays and footprinting experiments. Under multiple-turnover conditions Stop 225 exhibits biphasic kinetic behavior with both OG:A and G:A substrates, likely due to rate-limiting DNA product release. However, the rate of turnover of Stop 225 was increased 2-fold with OG:A substrates compared to the wild-type enzyme. In contrast, the intrinsic rate for adenine removal by Stop 225 from both G:A and OG:A substrates is significantly reduced (10- to 25-fold) compared to the wild-type. The affinity of Stop 225 for substrate analogs was dramatically reduced, as was the ability to discriminate between substrate analogs paired with OG over G. Interestingly, similar hydroxyl radical and DMS footprinting patterns are observed for Stop 225 and wild-type MutY bound to DNA duplexes containing OG opposite an abasic site mimic or a non-hydrogen bonding A analog, suggesting that similar regions of the DNA are contacted by both enzyme forms. Importantly, Stop 225 has a reduced ability to prevent DNA mutations in vivo. This implies that the reduced adenine glycosylase activity translates to a reduced capacity of Stop 225 to prevent DNA mutations in vivo.

Figure 1

Substrate, substrate analogs and product analogs for MutY. (A) Structures of an OG_syn:A_anti mispair based on NMR and X-ray crystallographic studies (51,55). (B) Structures of the 2′-deoxyadenosine analogs (F and FA), hydrophobic 2′-deoxyadenosine analog (M) and abasic site mimic (THF) used in this work.

Figure 2

A storage phosphor autoradiogram from a single turnover adenine glycosylase assay for MutY. A 30 bp duplex containing a central OG:A mismatch (with A-containing strand 5′-³²P-end-labeled) was incubated with wild-type MutY or Stop 225. The reaction was quenched and the abasic site product was cleaved upon addition of NaOH. The substrate (30 nt strand) was resolved from the product (14 nt strand) by denaturing PAGE. The control lane, with no enzyme added, is marked C. A description of the analysis used to determine rate constants is described in the text and the results are listed in Table 1.

Figure 3

Kinetic properties of Stop 225 with OG:A- and G:A-containing duplexes under multiple turnover conditions ([DNA] > [Stop 225]). Experiments were performed using 20 nM duplex DNA substrate containing either a G:A (closed circles) or OG:A (open circles) mismatch and Stop 225 (10 nM by Bradford determination). Based on the amplitude of the burst with the OG:A duplex, the active site concentration of Stop 225 was 2.1 nM. The k₃ values for these particular experiments were 0.03 and 0.007 min^–1 for the G:A and OG:A duplexes, respectively.

Figure 4

Storage phosphor autoradiogram of hydroxyl radical footprinting using MPE-Fe(II) of Stop 225 and wild-type MutY with an OG:THF-containing DNA duplex. Lanes 1–3, DNA, MPE-Fe(II) and enzyme at 100, 200 and 300 (wild-type) or 60, 120 and 180 nM (Stop 225), respectively; lane 4, DNA and MPE-Fe(II); lane 5, Maxam–Gilbert G+A reaction; lane 6, DNA only. The ³²P-end-labeled strand is indicated with an asterisk (*). The location of the OG or THF is indicated by an arrow. It should be noted that the active concentration of Stop 225 used in this experiment was less than that of wild-type MutY and therefore a lesser degree of protection was observed.

Figure 5

Histograms of hydroxyl radical and DMS footprinting experiments of Stop 225 and wild-type MutY with OG:M- and OG:THF-containing DNA duplexes. The duplex sequences containing either OG:M (A and B) and OG:THF (C and D) are shown with a histogram indicating the relative protection from hydroxyl radical cleavage for wild-type MutY (A and C) and Stop 225 MutY (B and D). The G residue shown to be hyper-reactive to DMS footprinting is shown in red and underlined. The data were obtained by quantitation of the storage phosphor autoradiograms from at least three separate experiments. The heights of the bars indicate the relative protection from hydroxyl radical cleavage. Both wild-type and Stop 225 MutY produce larger extents of protection of the OG:THF duplex than the OG:M duplex, therefore, the heights of the bars for the OG:THF duplex have been decreased by a factor of two. The observed protection patterns are strikingly similar for Stop 225 compared to the wild-type.

Scheme 1.