Abstract
DNA double-strand breaks (DSBs) can be repaired by one of two major pathways—non-homologous end-joining (NHEJ) and homologous recombination (HR)—depending on whether cells are in G1 or S/G2 phase, respectively. However, the mechanisms of DSB repair during M phase remain largely unclear. In this study, we demonstrate that transient treatment of M-phase cells with the chemotherapeutic topoisomerase inhibitor etoposide induced DSBs that were often associated with anaphase bridge formation and genome instability such as dicentric chromosomes. Although most of the DSBs were carried over into the next G1 phase, some were repaired during M phase. Both NHEJ and HR, in particular NHEJ, promoted anaphase-bridge formation, suggesting that these repair pathways can induce genome instability during M phase. On the other hand, C-terminal-binding protein interacting protein (CtIP) suppressed anaphase bridge formation, implying that CtIP function prevents genome instability during mitosis. We also observed M-phase-specific phosphorylation of XRCC4, a regulatory subunit of the ligase IV complex specialized for NHEJ. This phosphorylation required cyclin-dependent kinase (CDK) activity as well as polo-like kinase 1 (Plk1). A phosphorylation-defective XRCC4 mutant showed more efficient M-phase DSB repair accompanied with an increase in anaphase bridge formation. These results suggest that phosphorylation of XRCC4 suppresses DSB repair by modulating ligase IV function to prevent genome instability during M phase. Taken together, our results indicate that XRCC4 is required not only for the promotion of NHEJ during interphase but also for its M-phase-specific suppression of DSB repair.
Highlights
Double-strand breaks (DSBs) are one of the most consequential types of DNA damage
Inappropriate choice of the double-strand breaks (DSBs) repair pathway often results in perturbation or failure of DSB repair, which is occasionally associated with tumorigenesis
The DSB repair pathways in the cell-cycle phases G1, S, and G2 are well elucidated, little is known about how cells deal with DSBs induced during M phase
Summary
Double-strand breaks (DSBs) are one of the most consequential types of DNA damage. DSBs are usually repaired by one of two main repair pathways—canonical non-homologous end joining (C-NHEJ) or homologous recombination (HR) [1,2]. The protein mediator of DNA damage checkpoint 1 (MDC1) recognizes cH2AX and is phosphorylated by ATM [10]. Phosphorylated MDC1 recruits the RING finger (RNF)-containing E3 ubiquitin ligase RNF8, which mediates ubiquitination of proteins at the damage site Another E3 ubiquitin ligase, RNF168, recognizes RNF8 ubiquitination products and ubiquitinates additional proteins. This ubiquitination cascade leads to the recruitment of two main effector proteins, BRCA1 (breast cancer 1, early onset) and 53BP1 (p53-binding protein 1) to the DSB sites [11,12,13,14,15]. Subsequent recruitment of the single-stranded DNA binding protein, replication protein-A allows assembly of Rad recombinase filaments by Rad mediators such as BRCA2 and Rad paralogs including XRCC3 (X-ray repair cross-complementing group 3) to facilitate HR [2,18,19,20]
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