Structural Characterization of a Mouse Ortholog of Human NEIL3 with a Marked Preference for Single-Stranded DNA

Endonuclease VIII-like 3 (Neil3) is a DNA glycosylase of the base excision repair pathway that protects cells from oxidative DNA damage by excising a broad spectrum of cytotoxic and mutagenic base lesions. Interestingly, Neil3 exhibits an unusual preference for DNA with single-stranded regions. Here, we report the 2.0 angstrom crystal structure of a Neil3 enzyme. Although the glycosylase region of mouse Neil3 (MmuNeil3 Delta 324) exhibits the same overall fold as that of other Fpg/Nei proteins, it presents distinct structural features. First, MmuNeil3 Delta 324 lacks the alpha F-beta 9/10 loop that caps the flipped-out 8-oxoG in bacterial Fpg, which is consistent with its inability to cleave 8-oxoguanine. Second, Neil3 not only lacks two of the three void-filling residues that stabilize the opposite strand, but it also harbors negatively charged residues that create an unfavorable electrostatic environment for the phosphate backbone of that strand. These structural features provide insight into the substrate specificity and marked preference of Neil3 for ssDNA.