Abstract
Base excision repair is the major pathway for removal of oxidative DNA base damage. This pathway is initiated by DNA glycosylases, which recognize and excise damaged bases from DNA. In this work, we have purified the glycosylase domain (GD) of human DNA glycosylase NEIL3. The substrate specificity has been characterized and we have elucidated the catalytic mechanisms. GD NEIL3 excised the hydantoin lesions spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh) in single-stranded (ss) and double-stranded (ds) DNA efficiently. NEIL3 also removed 5-hydroxy-2'-deoxycytidine (5OHC) and 5-hydroxy-2'-deoxyuridine (5OHU) in ssDNA, but less efficiently than hydantoins. Unlike NEIL1 and NEIL2, which possess a β,δ-elimination activity, NEIL3 mainly incised damaged DNA by β-elimination. Further, the base excision and strand incision activities of NEIL3 exhibited a non-concerted action, indicating that NEIL3 mainly operate as a monofunctional DNA glycosylase. The site-specific NEIL3 mutant V2P, however, showed a concerted action, suggesting that the N-terminal amino group in Val2 is critical for the monofunctional modus. Finally, we demonstrated that residue Lys81 is essential for catalysis.
Highlights
The base excision repair pathway (BER) is the major pathway for repair of DNA damage caused by reactive oxygen species (ROS) [1, 2]
Liu and colleagues reported that mouse Neil3 and human NEIL3 removes oxidized purines and incises DNA by uncoupled DNA glycosylase/AP lyase activities, where base release is more efficient than strand incision [8, 9]
Neil2 is present in chordates and in the echinoderm sea urchin Strongylocentrotus purpuratus while Neil3 has only been found in chordates in previous studies
Summary
The base excision repair pathway (BER) is the major pathway for repair of DNA damage caused by reactive oxygen species (ROS) [1, 2]. DNA-glycosylase that excises the damaged base by cleavage of the N-glycosylic bond, creating an abasic (AP) site. A bifunctional glycosylase has an intrinsic lyase activity and incises the DNA strand 3’ to the AP site by either βelimination or β,δ-elimination. Takao and coworkers found a weak lyase activity for human NEIL3 on AP sites in ssDNA [7]. Liu and colleagues reported that mouse Neil and human NEIL3 removes oxidized purines and incises DNA by uncoupled DNA glycosylase/AP lyase activities, where base release is more efficient than strand incision [8, 9]. NEIL3, demonstrating that the enzyme mainly acts as a monofunctional DNA glycosylase with high affinity for the hydantoins Gh and Sp. Site-specific mutagenesis experiments show that Val is important for the non-concerted action of NEIL3, and Lys is essential for the catalytic activity
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