Duplex interrogation by a direct DNA repair protein in search of base damage

Abstract

ALKBH2 is a direct DNA repair dioxygenase guarding the mammalian genome against N(1)-methyladenine, N(3)-methylcytosine and 1,N(6)-ethenoadenine damage. A prerequisite for repair is to identify these lesions in the genome. Here we present crystal structures of human ALKBH2 bound to different duplex DNAs. Together with computational and biochemical analyses, our results suggest that DNA interrogation by ALKBH2 has two previously unknown features: (i) ALKBH2 probes base-pair stability and detects base pairs with reduced stability, and (ii) ALKBH2 does not have nor need a damage-checking site, which is critical for preventing spurious base cleavage for several glycosylases. The demethylation mechanism of ALKBH2 insures that only cognate lesions are oxidized and reversed to normal bases, and that a flipped, non-substrate base remains intact in the active site. Overall, the combination of duplex interrogation and oxidation chemistry allows ALKBH2 to detect and process diverse lesions efficiently and correctly.

Figure 1

Base pairs with different stability are discernible by ALKBH2. (a) Cartoon of the CG structure. (b) Local view showing the interrogation of the target C8:G8′ pair by ALKBH2, with residues Val101 and Phe102 highlighted. (c) Overall view of the AT structure. ALKBH2 is shown in green, DNA in yellow-orange, DNA bases from the upper strand in light magenta and those from the bottom strand in cyan.

Figure 2

Contributions of ALKBH2 to base-flipping in duplex DNA. (a) Cartoon view of the CI structure. The same color coding in Fig. 1 is used. (b) Computational analysis of free energy difference for ALKBH2 to break a C:G or C:I pair. States observed in crystal structures are in the orange boxes and contributions to the free energy difference ΔΔG are plotted.

Figure 3

ALKBH2 probes the stability of a base pair to detect DNA damages. (a) Side view of the CG structure. The approximate location of a flipped base is indicated with a magenta box and that of the orphaned base (which could have multiple conformations) is indicated with a dashed cyan box. (b) Overlay of the 1-meA structure (3BTY) and the CG structure. A clear shift of the hairpin loop is highlighted. The protein portion of 3BTY is shown in cyan and the DNA part is omitted for clarity purpose.

Figure 4

ALKBH2 does not have nor need a damage-checking site; its oxidation chemistry insures that non-substrate bases are not modified. (a) Superposition of the AT structure, εA structure, and 3BTY to show that the three flipped bases, damaged or not, are bound to the same site of ALKBH2. The view on the left panel is of the same angle as in Fig. 3; a 90° clockwise rotation of the left panel gives the view on the right. (b) Overlay of the CI and 3-meC structures using the protein part of the complex. A zoom-in view on the right shows the four active site residues that interact with the 3-meC base (Tyr122 and Glu175 form hydrogen-bonds to N4 of 3-meC; Phe124 and His171 stack against 3-meC). (c) Final positions of 3-meC (light magenta) and cytosine (pale cyan) in the active site of ALKBH2 are the same. The identical view as in b is shown, and all protein residues are omitted for clarity purpose. (d) Stereo view (which is a 90° clockwise rotation of c) of the metal site of the 3-meC structure. The aberrant methyl group (highlighted with yellow background color) is precisely positioned by ALKBH2 for efficient oxidation. The approximate location of the putative iron(IV)-oxo species, after 2-ketoglutarate is converted to succinate, is colored in blue.