Hypersensitivity of mouse NEIL1-knockdown cells to hydrogen peroxide during S phase

Abstract

Oxidative base damage occurs spontaneously due to reactive oxygen species generated as byproducts of respiration and other pathological processes in mammalian cells. Many oxidized bases are mutagenic and/or toxic, and most are repaired through the base excision repair pathway. Human endonuclease VIII-like protein 1 (hNEIL1) is thought to play an important role during the S phase of the cell cycle by removing oxidized bases in DNA replication fork-like (bubble) structures, and the protein level of hNEIL1 is increased in S phase. Compared with hNEIL1, there is relatively little information on the properties of the mouse ortholog mNEIL1. Since mouse cell nuclei lack endonuclease III-like protein (NTH) activity, in contrast to human cell nuclei, mNEIL1 is a major DNA glycosylase for repair of oxidized pyrimidines in mouse nuclei. In this study, we made mNEIL1-knockdown cells using an shRNA expression vector and examined the cell cycle-related variation in hydrogen peroxide (H2O2) sensitivity. Hypersensitivity to H2O2 caused by mNEIL1 knockdown was more significant in S phase than in G1 phase, suggesting that mNEIL1 has an important role during S phase, similarly to hNEIL1.

Ubiquitylation-dependent regulation of NEIL1 by Mule and TRIM26 is required for the cellular DNA damage response

“…However a knockdown of TRIM26, or a partial NEIL1 overexpression, does mediate resistance of cells to IR. This is consistent with previously published data demonstrating that mouse embryonic cells lacking NEIL1 are hypersensitive to IR and hydrogen peroxide-induced cell death ( 15 , 16 ). As we revealed cellular resistance by clonogenic assays, in which colonies are allowed to grow for ∼7 days post-irradiation, this would suggest that NEIL1 acts on specific DNA damage substrates (e.g.…”

“…Since NEIL1 has been demonstrated to be an important enzyme in removal of oxidised DNA bases, particularly in single stranded DNA generated through transcription and replication ( 12 , 13 ), and is therefore important in the cellular response to IR and oxidative stress ( 15 , 16 ), we hypothesized that the protein should be under the control of the UPP catalysed by E3 ubiquitin ligases. Therefore, using an in vitro ubiquitylation assay in combination with cell extract fractionation by column chromatography (Figure 1A ), an approach which we have used successfully for other BER proteins ( 28 , 31 , 32 ), we purified the major E3 ubiquitin ligases for NEIL1.…”

“…NEIL1 plays an important role in suppressing the accumulation of DNA damage, and thus in maintaining genome stability, by repairing oxidised DNA base damage. Indeed, this is highlighted by observations that NEIL1 protects cells from hydrogen peroxide and IR-induced cell death ( 15 , 16 ). Furthermore, a deficiency of NEIL1 in knockout mouse models displays an obese phenotype associated with a metabolic syndrome ( 17 , 18 ), and loss of NEIL1 has been associated with the earliest symptoms of age-related neurodegenerative disorders such as Alzheimer's and Parkinson's diseases in humans ( 20 , 21 ).…”

“…Further data have also implicated the involvement of NEIL1 specifically in a replication-associated DNA repair role ( 14 ). The importance of NEIL1 in the cellular response to DNA damage has been shown by the observation that NEIL1 protects cells from hydrogen peroxide and IR-induced cell death ( 15 , 16 ). Interestingly, a deficiency of NEIL1 in knockout mouse models displays an obese phenotype associated with a metabolic syndrome ( 17 , 18 ).…”

Distinct Mechanisms of Target Search by Endonuclease VIII-like DNA Glycosylases

“…Despite a number of studies characterizing their biochemistry, it is still an open question what is their main role in the cell. Thus far, it has been shown that NEIL1 interacts with some replication proteins, is up-regulated in the S phase and, therefore, likely repairs damaged bases in double-stranded (ds) DNA during replication [ 27 , 33 , 34 , 35 , 36 , 37 ]. NEIL2 prefers bubble structures and single-stranded (ss) DNA, interacts with RNA polymerase II and the transcriptional regulator heterogeneous nuclear ribonucleoprotein-U and has been suggested to repair DNA lesions during transcription [ 38 , 39 , 40 , 41 ].…”