Abstract
ABSTRACTEndonuclease III-like protein 1 (NTH1) is a DNA glycosylase required for the repair of oxidized bases, such as thymine glycol, within the base excision repair pathway. We examined regulation of NTH1 protein by the ubiquitin proteasome pathway and identified the E3 ubiquitin ligase tripartite motif 26 (TRIM26) as the major enzyme targeting NTH1 for polyubiquitylation. We demonstrate that TRIM26 catalyzes ubiquitylation of NTH1 predominantly on lysine 67 present within the N terminus of the protein in vitro. In addition, the stability of a ubiquitylation-deficient protein mutant of NTH1 (lysine to arginine) at this specific residue was significantly increased in comparison to the wild-type protein when transiently expressed in cultured cells. We also demonstrate that cellular NTH1 protein is induced in response to oxidative stress following hydrogen peroxide treatment of cells and that accumulation of NTH1 on chromatin is exacerbated in the absence of TRIM26 through small interfering RNA (siRNA) depletion. Stabilization of NTH1 following TRIM26 siRNA also causes significant acceleration in the kinetics of DNA damage repair and cellular resistance to oxidative stress, which can be recapitulated by moderate overexpression of NTH1. This demonstrates the importance of TRIM26 in regulating the cellular levels of NTH1, particularly under conditions of oxidative stress.
Highlights
Endonuclease III-like protein 1 (NTH1) is a DNA glycosylase required for the repair of oxidized bases, such as thymine glycol, within the base excision repair pathway
We have demonstrated that two E3 ubiquitin ligases (E3s), Mcl-1 ubiquitin ligase E3 (Mule) and tripartite motif 26 (TRIM26), are the major cellular enzymes involved in the ubiquitylation-dependent degradation of NEIL1 and that this mechanism is important for modulating the cellular response to ionizing-radiation-induced DNA damage [7]
The base excision repair (BER) pathway plays a major role in suppressing the accumulation of oxidative DNA damage, and NTH1 is a specific DNA glycosylase that recognizes and excises oxidized pyrimidines, including 5-hydroxyuracil, 5-hydrocytosine, and thymine glycol, from DNA
Summary
Endonuclease III-like protein 1 (NTH1) is a DNA glycosylase required for the repair of oxidized bases, such as thymine glycol, within the base excision repair pathway. Small interfering RNA (siRNA) depletion of nth in TK6 cells causes sensitivity to hydrogen peroxide treatment, whereas cellular resistance was observed following overexpression of NTH1 [11] These data demonstrate the importance of regulating NTH1 protein levels in the cellular response to oxidative stress. Expression of a natural single nucleotide polymorphism of NTH1 (D239Y), identified in approximately 6% of the population, causes sensitivity to oxidative stress, causes accumulation of DNA double-strand breaks and chromosomal aberrations, and induces cellular transformation [16] These studies demonstrate the importance of NTH1, the maintenance of the cellular protein levels of NTH1, in conserving genome stability. We report the purification of TRIM26 and its identification as the major E3 that regulates ubiquitylation-dependent degradation of NTH1 in vitro and in vivo and that this mechanism is important in controlling the cellular response to oxidative stress
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