ATR- and ATM-Mediated DNA Damage Response Is Dependent on Excision Repair Assembly during G1 but Not in S Phase of Cell Cycle.

Alo Ray,Gulzar Wani,Chessica Blevins,Altaf A Wani,Michael Shing-Yan Huen

doi:10.1371/journal.pone.0159344

Abstract

Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation.

Highlights

In response to DNA damage, living cells arrest at discrete phases of the cell cycle either to allow DNA repair which is essential for cell survival or if the damage is too high promote cell death [1;2]
H2AX phosphorylation is evident at the same ten minute post-ultraviolet radiation (UVR) exposure time in OSU-2 cells (Fig 1C). These results indicate that ATR and ATM recruitment and H2AX phosphorylation start early upon UV irradiation and these proteins co-localize with early damage recognition factor XPC
We have previously demonstrated that ATR and ATM recruitment is dependent on the assembly of functional DNA repair proteins, and in the present study we have established that the UVR-mediated ATR and ATM recruitment occurs as a very early event in asynchronous and G1 arrested cells (Figs 1 & 3)

Summary

Introduction

In response to DNA damage, living cells arrest at discrete phases of the cell cycle either to allow DNA repair which is essential for cell survival or if the damage is too high promote cell death [1;2]. The mammalian nucleotide excision repair (NER) pathway removes a wide range of chemically and conformationally diverse DNA adducts, including ultraviolet radiation (UVR)-induced bulky DNA adducts, e.g., cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproducts (6-4PP) [3]. In GG-NER, damage is recognized by the DDB (damaged DNA binding protein), involving DDB1 and DDB2, and XPC (Xeroderma pigmentosum complementation group C)-RAD23B complexes [5;6]. The DDB1-CUL4ROC1 complex associates with DDB2 adapter and Cullin 4A-mediated proteolysis of DDB2 at the DNA damage sites regulates the lesion recognition by XPC [9]. XPC orchestrates the sequential recruiting of factors of multi-protein NER complex including XPA, XPG, and TFIIH components that enable opening of the DNA helix around the damage site to form a bubble [7]. XPA is an integral component of DNA damage processing by both GG-NER and TC-NER

Methods

Results

Conclusion