Cell cycle-dependent phosphorylation regulates RECQL4 pathway choice and ubiquitination in DNA double-strand break repair

Huiming Lu,Priscella P Holland,Jane Tian,Deborah L Croteau,Prabhat Khadka,Mustafa Okur,Raghavendra A Shamanna,Tomasz Kulikowicz,Vilhelm A Bohr,Jessica K De Freitas,Anthony J Davis

doi:10.1038/s41467-017-02146-3

Abstract

Pathway choice within DNA double-strand break (DSB) repair is a tightly regulated process to maintain genome integrity. RECQL4, deficient in Rothmund-Thomson Syndrome, promotes the two major DSB repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). Here we report that RECQL4 promotes and coordinates NHEJ and HR in different cell cycle phases. RECQL4 interacts with Ku70 to promote NHEJ in G1 when overall cyclin-dependent kinase (CDK) activity is low. During S/G2 phases, CDK1 and CDK2 (CDK1/2) phosphorylate RECQL4 on serines 89 and 251, enhancing MRE11/RECQL4 interaction and RECQL4 recruitment to DSBs. After phosphorylation, RECQL4 is ubiquitinated by the DDB1-CUL4A E3 ubiquitin ligase, which facilitates its accumulation at DSBs. Phosphorylation of RECQL4 stimulates its helicase activity, promotes DNA end resection, increases HR and cell survival after ionizing radiation, and prevents cellular senescence. Collectively, we propose that RECQL4 modulates the pathway choice of NHEJ and HR in a cell cycle-dependent manner.

Highlights

DNA double-strand breaks (DSBs) are generated by endogenous stress, during programmed recombination events, or after exposure to exogenous sources, such as ionizing radiation (IR) and chemotherapeutics[1]
In DR-GFP U2OS cells, I-SceI endonuclease generates a DSB in the cassette of the dysfunctional GFP gene, which can be repaired via homologous recombination (HR) using the downstream GFP fragment (Fig. 1a)[35]
We report here that CDK1/2-mediated phosphorylation and DDB1-CUL4Adependent ubiquitination of RECQL4 regulate DSB repair pathway choice in a cell cycle-dependent manner

Summary

Introduction

DNA double-strand breaks (DSBs) are generated by endogenous stress, during programmed recombination events, or after exposure to exogenous sources, such as ionizing radiation (IR) and chemotherapeutics[1]. DSB repair pathway choice is a tightly regulated process that is influenced by many factors, including the cell cycle phase, DNA end resection, and post-translational modifications[4]. E3 ubiquitin ligase, a member of the cullin-RING E3 ubiquitin ligase family, regulates cell cycle progression, DNA replication, and genome integrity[20] Both core components, DDB1 and CUL4A, are recruited to laser-induced DSBs, and the DDB1CUL4 E3 ubiquitin ligase promotes DNA end resection and HR by regulating histone protein monoubiquitination[21,22]. We report a cell cycledependent regulation of RECQL4 in DSB repair pathway choice, driven by its CDK1/2-mediated phosphorylation and DDB1-CUL4A-dependent ubiquitination in S/G2 phases of the cell cycle

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Results

Conclusion