Serines 440 and 467 in the Werner syndrome protein are phosphorylated by DNA-PK and affects its dynamics in response to DNA double strand breaks

Rika Kusumoto-Matsuo,Dale Ramsden,Parimal Karmakar,Alfred May,Vilhelm A Bohr,Deblina Ghosh

doi:10.18632/aging.100629

Abstract

WRN protein, defective in Werner syndrome (WS), a human segmental progeria, is a target of serine/threonine kinases involved in sensing DNA damage. DNA-PK phosphorylates WRN in response to DNA double strand breaks (DSBs). However, the main phosphorylation sites and functional importance of the phosphorylation of WRN has remained unclear. Here, we identify Ser-440 and -467 in WRN as major phosphorylation sites mediated by DNA-PK.In vitro, DNA-PK fails to phosphorylate a GST-WRN fragment with S440A and/or S467A substitution. In addition, full length WRN with the mutation expressed in 293T cells was not phosphorylated in response to DSBs produced by bleomycin. Accumulation of the mutant WRN at the site of laser-induced DSBs occurred with the same kinetics as wild type WRN in live HeLa cells. While the wild type WRN relocalized to the nucleoli after 24 hours recovery from etoposide-induced DSBs, the mutant WRN remained mostly in the nucleoplasm. Consistent with this, WS cells expressing the mutants exhibited less DNA repair efficiency and more sensitivity to etoposide, compared to those expressing wild type. Our findings indicate that phosphorylation of Ser-440 and -467 in WRN are important for relocalization of WRN to nucleoli, and that it is required for efficient DSB repair.

Highlights

Werner Syndrome (WS) is an autosomal recessive disorder characterized by premature aging, elevated genomic instability and increased cancer [1]
We found that more double strand breaks (DSBs) persisted in WS cells expressing WRN mutants compared to wild type after 24 hour-recovery from etoposide exposure
The WS cells expressing EGFP-WRN showed decreased sensitivity to etoposide, compared to those transfected with empty vector (Fig. 6), consistent with a previous report[24], though inconsistent results have been reported about sensitivity of WS cells to etoposide

Summary

Introduction

Werner Syndrome (WS) is an autosomal recessive disorder characterized by premature aging, elevated genomic instability and increased cancer [1]. WS cells exhibit genomic instability with DNA deletions, insertions, and rearrangements [3]. WS cells show hypersensitivity to some DNA damaging agents, including 4-nitroquinoline-1-oxide (4NQO) and topoisomerase inhibitors as well as mild sensitivity to ionizing radiation [4, 5]. WRN localizes to nucleoli under normal conditions, and translocates to the nucleoplasm in response to stress. It is proposed that post-translational modifications of WRN such as phosphorylation, acetylation, and sumoylation occur in response to stresses and affect its dynamics [8]. WRN is phosphorylated at serine, threonine or tyrosine residues by several kinases in response to DNA damage or stress.

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Results

Conclusion