Acetylation regulates ribonucleotide reductase activity and cancer cell growth

Guo Chen,Youwei Sun,Paul W Doetsch,Yin Luo,Walter J Curran,Madhusmita Behera,Xingming Deng,Kurt Warncke,Duc M Duong,Haian Fu,David S Yu,Suresh S Ramalingam,Michael Lammers

doi:10.1038/s41467-019-11214-9

Abstract

Ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleoside diphosphates (dNDPs) to provide dNTP precursors for DNA synthesis. Here, we report that acetylation and deacetylation of the RRM2 subunit of RNR acts as a molecular switch that impacts RNR activity, dNTP synthesis, and DNA replication fork progression. Acetylation of RRM2 at K95 abrogates RNR activity by disrupting its homodimer assembly. RRM2 is directly acetylated by KAT7, and deacetylated by Sirt2, respectively. Sirt2, which level peak in S phase, sustains RNR activity at or above a threshold level required for dNTPs synthesis. We also find that radiation or camptothecin-induced DNA damage promotes RRM2 deacetylation by enhancing Sirt2–RRM2 interaction. Acetylation of RRM2 at K95 results in the reduction of the dNTP pool, DNA replication fork stalling, and the suppression of tumor cell growth in vitro and in vivo. This study therefore identifies acetylation as a regulatory mechanism governing RNR activity.

Highlights

Ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleoside diphosphates to provide dNTP precursors for DNA synthesis
To examine whether protein acetylation is involved in the regulation of dNTP synthesis, human lung cancer H1299 cells were treated with two different deacetylase inhibitors, trichostatin A (TSA) that inhibits histone deacetylase (HDAC) classes I, II, and IV, and nicotinamide (NAM) that inhibits sirtuin family members[24], alone or in combination, leading to increased global lysine acetylation in cells
Combined treatment with TSA and NAM resulted in significantly decreased levels of all four dNTPs (Supplementary Fig. 1b, c), indicating that the dNTP synthesis pathway may be disturbed by protein acetylation

Summary

Introduction

Ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleoside diphosphates (dNDPs) to provide dNTP precursors for DNA synthesis. We report that acetylation and deacetylation of the RRM2 subunit of RNR acts as a molecular switch that impacts RNR activity, dNTP synthesis, and DNA replication fork progression. Acetylation of RRM2 at K95 results in the reduction of the dNTP pool, DNA replication fork stalling, and the suppression of tumor cell growth in vitro and in vivo. Several therapeutic RNR inhibitors, including gemcitabine, clofarabine, and hydroxyurea, are employed clinically to treat a number of cancers[16,17] Such small molecule RNR inhibitors fall into two classes: nucleoside analogs and redox-active metal chelators, which target RRM1 and RRM2, respectively[14]. Reversible lysine acetylation/deacetylation plays a critical role in regulating many essential proteins involved in diverse cellular processes, including DNA repair, chromatin remodeling, transcription, metabolism, cell survival, and proliferation[18,19]. RRM2 acetylation at K95 suppresses tumor cell growth in vitro and in vivo, and is a potentially attractive strategy for cancer therapy

Methods

Results

Conclusion