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

Abstract

Summary Endonuclease VIII-like 3 (Neil3) is a DNA glycosylase of the base excision repair pathway which 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 first crystal structure of a Neil3 enzyme. Although the glycosylase region of mouse Neil3 (MmuNeil3Δ324) exhibits the same overall fold as that of other Fpg/Nei proteins, it presents distinct structural features. First, MmuNeil3Δ324 lacks the “αF-β9/10 loop” that caps the flipped-out 8-oxoG in bacterial Fpg, which is consistent with its inability to cleave 8-oxoguanine. Secondly, Neil3 not only lacks two of the three void-filling residues that stabilize the opposite strand but it also harbors negatively-charged residues which create an unfavorable electrostatic environment for the phosphate backbone of that strand. These structural features provide insight into Neil3's substrate specificity and marked preference for ssDNA.

Neil3 and NEIL1 DNA Glycosylases Remove Oxidative Damages from Quadruplex DNA and Exhibit Preferences for Lesions in the Telomeric Sequence Context

“…First, the extents of cleavage are greater for each single-stranded representative by comparison with its double-stranded mate. This is consistent with the possibility that availability of Tg for insertion into the active site pocket is greater in the single-stranded substrates due to the absence of the opposite strand and is in keeping with our prior biochemical studies of Neil3 showing its preference for lesions in singlestranded substrates (13) and with its structure (63) showing why. Similarly, there is a strong effect of context, that is, the extents of cleavage are greater for each RSC context by comparison with its R mate.…”

“…It turns out that NEIL1, similar to most members of the Fpg/Nei family, contains three void-filling residues that help extrude the lesion from the DNA into the active site pocket, whereas two of them are missing in mNeil3 (63). Although NEIL1 has no preference for Tg in the telomere sequence context, it did show a slight preference for Gh (Fig.…”

Deficiency of base excision repair enzyme NEIL3 drives increased predisposition to autoimmunity

“…NEIL3 is an atypical glycosylase with an N-terminal glycosylase domain and a disordered C-terminal domain with unknown function. The glycosylase domain of NEIL3 preferentially removes oxidized bases in single-stranded DNA, and the optimal substrates for NEIL3 are spiroiminodihydantoin (SP) and guanidinohydantoin (Gh), further oxidation products of 8-oxo-7,8-dihydroguanine (8-oxoG) (36)(37)(38). NEIL3 transcripts are expressed in human thymus and testes (39,40) and at high levels in tumor samples (41,42).…”

“…There was no increase in the levels of autoantibodies in sera from the patients' parents and unaffected siblings (data not shown). We identified in the second ROH 2 adjacent coding variants in NEIL3, which codes for nei endonuclease VIII-like 3 enzyme (36)(37)(38). In addition, we found variants in the coding sequences of 3 other genes outside the 2 ROH: KCNA10 (codes for potassium channel, voltage-gated shaker related subfamily A, member 10), ST7L (codes for suppression of tumorigenicity 7-like protein), and ACTR1B (codes for actin-related protein 1 homolog B).…”

“…The crystal structure of the unliganded mouse ortholog of human NEIL3 (mNEIL3) showed that D133 in mNEIL3, the equivalent to the mutated D132 in the patients' NEIL3, is located in the DNA-binding pocket of the enzyme (38), suggesting that the D132 residue may be important for NEIL3 activity. To test this hypothesis, full-length WT and D132V NEIL3 were expressed in HeLa cells and immunoprecipitated, and their glycosylase activity was determined by immunoprecipitation-coupled glycosylase activity assay (47).…”