The E3 Ubiquitin Ligase NEDD4L Targets OGG1 for Ubiquitylation and Modulates the Cellular DNA Damage Response.

Jonathan R Hughes,Jason L Parsons

doi:10.3389/fcell.2020.607060

Abstract

8-Oxoguanine DNA glycosylase (OGG1) is the major cellular enzyme required for the excision of 8-oxoguanine DNA base lesions in DNA through the base excision repair (BER) pathway, and therefore plays a major role in suppressing mutagenesis and in controlling genome stability. However, the mechanism of regulation of cellular OGG1 protein, particularly in response to oxidative stress, is unclear. We have purified the major E3 ubiquitin ligase responsible for OGG1 ubiquitylation from human cell extracts, and identify this as E3 ubiquitin-protein ligase NEDD4-like (NEDD4L). We demonstrate that recombinant NEDD4L stimulates ubiquitylation of OGG1 in vitro, particularly on lysine 341, and that NEDD4L and OGG1 interact in U2OS cells. Depletion of NEDD4L in U2OS cells has no impact on the stability and steady-state protein levels of OGG1, however, OGG1 stability is enhanced in response to oxidative stress induced by ionizing radiation. Furthermore, ubiquitylation of OGG1 by NEDD4L in vitro inhibits its DNA glycosylase/lyase activity. As a consequence of prolonged OGG1 stability and increased excision activity in the absence of NEDD4L, cells display increased DNA repair capacity but conversely that this decreases cell survival post-irradiation. This effect can be reproduced following OGG1 overexpression, suggesting that dysregulation of OGG1 increases the formation of lethal intermediate DNA lesions. Our study therefore highlights the importance of balancing OGG1 protein levels and BER capacity in maintaining genome stability.

Highlights

Reactive oxygen species (ROS) are produced in cells as a consequence of oxidative metabolism, and in addition to exogenous sources including ionizing radiation (IR), these are able to attack DNA and disrupt genome integrity
Since we determined that lysine 341 is the major ubiquitylation site within OGG1 catalyzed by NEDD4L in vitro (Figure 3F), we examined the comparative stability of wild type and K341R OGG1 proteins following expression in U2OS cells (Figure 4F and Supplementary Figures 6I,J)
Whilst mice deficient in OGG1 only display moderate increases in spontaneous mutation rates associated with accumulation of 8-oxoguanine (Klungland et al, 1999; Minowa et al, 2000; Sakumi et al, 2003), altered OGG1 protein expression and activity has been observed in a number of different cancer cell types, including prostate, head and neck squamous cell carcinoma and colorectal cancer (Kondo et al, 2000; Trzeciak et al, 2004; Paz-Elizur et al, 2006; Kumar et al, 2012)

Summary

Introduction

Reactive oxygen species (ROS) are produced in cells as a consequence of oxidative metabolism, and in addition to exogenous sources including ionizing radiation (IR), these are able to attack DNA and disrupt genome integrity. Ubiquitylation of OGG1 by NEDD4L excise DNA base modifications (Jacobs and Schar, 2012;Wallace, 2013), as well as other protein members of the base excision repair (BER) pathway that restore the DNA to its undamaged form. These proteins include AP endonuclease 1 (APE1) that incises the AP site formed following DNA glycosylase incision, DNA polymerase β (Pol β) that inserts the correct nucleotide and removes the 5 -deoxyribosephosphate moiety, and DNA ligase IIIα-X-ray repair cross-complementing protein 1 (Lig IIIα-XRCC1) complex that seals the nick in the DNA backbone (Parsons and Dianov, 2013). It is clearly evident that BER proteins, and the pathway itself, is tightly regulated by ubiquitylation in response to the endogenous levels of DNA damage

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Conclusion