SUMOylation mediates CtIP’s functions in DNA end resection and replication fork protection

Abstract

Double-strand breaks and stalled replication forks are a significant threat to genomic stability that can lead to chromosomal rearrangements or cell death. The protein CtIP promotes DNA end resection, an early step in homologous recombination repair, and has been found to protect perturbed forks from excessive nucleolytic degradation. However, it remains unknown how CtIP’s function in fork protection is regulated. Here, we show that CtIP recruitment to sites of DNA damage and replication stress is impaired upon global inhibition of SUMOylation. We demonstrate that CtIP is a target for modification by SUMO-2 and that this occurs constitutively during S phase. The modification is dependent on the activities of cyclin-dependent kinases and the PI-3-kinase-related kinase ATR on CtIP’s carboxyl-terminal region, an interaction with the replication factor PCNA, and the E3 SUMO ligase PIAS4. We also identify residue K578 as a key residue that contributes to CtIP SUMOylation. Functionally, a CtIP mutant where K578 is substituted with a non-SUMOylatable arginine residue is defective in promoting DNA end resection, homologous recombination, and in protecting stalled replication forks from excessive nucleolytic degradation. Our results shed further light on the tightly coordinated regulation of CtIP by SUMOylation in the maintenance of genome stability.

Crosstalk between SUMOylation and ubiquitylation controls DNA end resection by maintaining MRE11 homeostasis on chromatin

“…In contrast, other SUMO E3 ligases did not lead to the same increase in MRE11 SUMOylation. PIAS4 and CBX4, which have been reported to be the E3 ligases for CtIP SUMOylation 26,27 , exerted only a minor effect on MRE11 SUMOylation (Fig. 2a, b).…”

TOPORS-mediated RAD51 SUMOylation facilitates homologous recombination repair

“…The primary SUMO E3 ligases involved are PIAS1 and PIAS4, which themselves localize to laser microirradiation-induced DSBs, to promote DSB repair in part by inducing SUMOylation of 53BP1 ( 43 ) and BRCA1 ( 43 , 50 ). PIAS4 is also required for proper regulation of the HR repair, through SUMOylating the DSB end resection nuclease CtIP ( 51 , 52 ). Consistently, PIAS4 is required for RAD51 foci formation ( 35 ); this study did not find RAD51 itself to be SUMOylated, but a SIM in RAD51 is required for HR repair, suggesting that a noncovalent SIM–SUMO interaction is involved in regulating the HR repair.…”

Cellular mechanism of action of 2-nitroimidazoles as hypoxia-selective therapeutic agents

“…The effect of drugs on DNA replication was measured by a DNA fibre assay as previously described with some modifications [ 21 ]. Briefly, cells were incubated with 5-iodo-2′-deoxyuridine (33 μM; IdU; I7756, Sigma-Aldrich) for 30 min followed by several PBS washes.…”