A phosphate binding pocket is a key determinant of exo- versus endo-nucleolytic activity in the SNM1 nuclease family

Abstract The SNM1 nucleases which help maintain genome integrity are members of the metallo-β-lactamase (MBL) structural superfamily. Their conserved MBL-β-CASP-fold SNM1 core provides a molecular scaffold forming an active site which coordinates the metal ions required for catalysis. The features that determine SNM1 endo- versus exonuclease activity, and which control substrate selectivity and binding are poorly understood. We describe a structure of SNM1B/Apollo with two nucleotides bound to its active site, resembling the product state of its exonuclease reaction. The structure enables definition of key SNM1B residues that form contacts with DNA and identifies a 5′ phosphate binding pocket, which we demonstrate is important in catalysis and which has a key role in determining endo- versus exonucleolytic activity across the SNM1 family. We probed the capacity of SNM1B to digest past sites of common endogenous DNA lesions and find that base modifications planar to the nucleobase can be accommodated due to the open architecture of the active site, but lesions axial to the plane of the nucleobase are not well tolerated due to constriction around the altered base. We propose that SNM1B/Apollo might employ its activity to help remove common oxidative lesions from telomeres.

. Generation of structurally diverse DNA substrates.
A. Single-stranded 51 nt oligonucleotides radiolabelled at the 3ʹ end were annealed with the appropriate corresponding oligonucleotide to generate the substrates shown in schematic form (see Suppl. Table 1 for DNA sequences). Red stars denote the α-32 P-dATP label. B. Non-denaturing PAGE demonstrating correct annealing of oligonucleotide substrates.
Suppl. Figure 4. Schematic representation of the real-time fluorescence-based nuclease assay. The intact DNA substrate (Suppl. Table 1) does not fluoresce, due to the proximity of the black-hole-quench (BHQ-1) and fluorescein groups. Following exonucleolytic digestion of SNM1B 1-355 past the fluorescein-conjugated-T, and subsequent uncoupling of the fluorescein and BHQ-1, there is a concomitant increase in fluorescence that can be measured. Suppl. Figure 6. The coordination network of the phosphate group of AMP_2 indicates residues likely involved in catalysis and / or binding the product. The active site metal ions (Ni 2+ at the M1 site, and Fe at the M2 site are as indicated). This is an expansion of Figure 1E.
Suppl. Figure 7. Electron density map of the active site of SNM1B 1-355 (nucleotide form).
A 2F o -1F c electron density map of WT SNM1B 1-355 nucleotide complex contoured at 1 sigma in the vicinity of the active site, with nucleotides in green and metals in pink.

Suppl. Figure 8. Metal ion co-ordination in the active site of SNM1B 1-355 (apo form).
The unliganded structure of SNM1B 1-355 (apo form) has only the first metal ion binding site (M1) occupied (modelled as a Ni 2+ ion). The metal ion coordinating residues are as labelled, the Ni 2+ ion is depicted as a green sphere, and the coordinating water molecule is a red sphere.
Suppl. Figure 9. Inductively coupled plasma mass spectrometry (ICP-MS) of WT SNM1B    Suppl. Figure 16. The nuclease activity of SNM1B 1-355 is inhibited by the addition of non-labelled excess ssDNA substrate. Gel based 'competition' assay whereby 100 nM of DNA substrate was added to 1.0 nM SNM1B 1-355 and incubated at 37 o C for 10 min, at which point the reaction was either stopped, or increasing amounts of unlabeled ssDNA substrate was added to the reaction mix, and then incubated for a total period of 60 min. A single 60 min reaction was included where no additional unlabeled substrate was added. Reaction products were analysed by 20% denaturing PAGE; each gel is representative of at least three individual experiments.
A. DNA oligonucleotide sequences and codes used to denote them. P = phosphate; OH = hydroxyl. B. The oligonucleotide sequences that are annealed to generate the indicated substrate structures. In the first column, the asterisk indicates the radiolabelled strand. In the 'substrate structure' column the red asterisk indicates the radiolabel; a blue circle an 8oxo-guanine; a green circle, other DNA damage lesions; a yellow circle a fluorescein; and a dark blue circle, a BHQ.