Expression and purification of active mouse and human NEIL3 proteins

Abstract

Endonuclease VIII-like 3 (Neil3) is one of the five DNA glycosylases found in mammals that recognize and remove oxidized bases, and initiate the Base Excision Repair (BER) pathway. Previous attempts to express and purify the mouse and human orthologs of Neil3 in their active form have not been successful. Here we report the construction of bicistronic expression vectors for expressing in Escherichia coli the full-length mouse Neil3 (MmuNeil3), its glycosylase domain (MmuNeil3Δ324), as well as the glycosylase domain of human Neil3 (NEIL3Δ324). The purified Neil3 proteins are all active, and NEIL3Δ324 exhibits similar glycosylase/lyase activity as MmuNeil3Δ324 on both single-stranded and double-stranded substrates containing thymine glycol (Tg), spiroiminodihydantoin (Sp) or an abasic site (AP). We show that N-terminal initiator methionine processing is critical for the activity of both mouse and human Neil3 proteins. Co-expressing an E. coli methionine aminopeptidase (EcoMap) Y168A variant with MmuNeil3, MmuNeil3Δ324 and NEIL3Δ324 improves the N-terminal methionine processing and increases the percentage of active Neil3 proteins in the preparation. The purified Neil3 proteins are suitable for biochemical, structural and functional studies.

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

“…Enzymes-Details of the cloning, expression, and purification of the glycosylase domain of the Mus musculus Neil3 were described previously (42). Unless it was specifically pointed out, the mNeil3 enzyme used in this study was the glycosylase domain of M. musculus Neil3 (M. musculus Neil3⌬324).…”

Formation and processing of DNA damage substrates for the hNEIL enzymes

“…During establishment of the nomenclature of the Neils, they have been referred to as hFPG [169], hNEI [170], and NEH1 [171], ultimately the name NEIL (Nei-Like) was suggested by the Human Genome Organization. NEIL1 and 2 are bifunctional glycosylases with β,δ elimination activity through the action of an N-terminal proline [169, 171–173], and NEIL3 is mainly a monofunctional glycosylase via an N-terminal valine residue [174, 175]. The remainder of this review will focus on DNA glycosylases of the BER pathway processing the hydantoin substrates Sp or Gh and the single report on 2Ih in many different structural contexts.…”

A genome-wide analysis of gene–caffeine consumption interaction on basal cell carcinoma

“…NEIL3 encodes a DNA glycosylase that recognizes and removes lesions produced by oxidative stress, such as spiroiminodihydantoin (Sp), guanidinohydantoin (Gh) and 8-oxoguanine (8-oxoG), primarily in single-stranded DNA (ssDNA) (31). NEIL3 has been shown to be an important facilitator of cell proliferation in neural stem/ progenitor cells and tumor cells, suggesting its possible role in replication-associated DNA repair (32,33). Some studies have reported that polymorphisms of DNA glycosylases may possess altered enzymatic activity, increasing the risk of inflammation-related cancers (34,35).…”