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

• Published in: Nucleic acids research Vol. 49; no. 2; pp. 928 - 953
• Main Authors: Locke, Andrew J, Hossain, Lazina, McCrostie, Glynnis, Ronato, Daryl A, Fitieh, Amira, Rafique, Tanzeem Ahmed, Mashayekhi, Fatemeh, Motamedi, Mobina, Masson, Jean-Yves, Ismail, Ismail Hassan
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 25-01-2021
• Subjects: Amino Acid Substitution; Arginine - chemistry; Ataxia Telangiectasia Mutated Proteins - metabolism; Cell Line; Cyclin-Dependent Kinases - metabolism; DNA Breaks, Double-Stranded; DNA End-Joining Repair - genetics; DNA End-Joining Repair - physiology; DNA Replication; Endodeoxyribonucleases - chemistry; Endodeoxyribonucleases - metabolism; Endodeoxyribonucleases - physiology; Genes, Reporter; Genome Integrity, Repair and; Genomic Instability; Humans; Lysine - chemistry; Poly-ADP-Ribose Binding Proteins - physiology; Proliferating Cell Nuclear Antigen - metabolism; Protein Inhibitors of Activated STAT - physiology; Protein Interaction Mapping; Protein Processing, Post-Translational; Recombinant Fusion Proteins - metabolism; Recombinational DNA Repair - genetics; Recombinational DNA Repair - physiology; RNA Interference; RNA, Small Interfering - genetics; RNA, Small Interfering - pharmacology; Sumoylation
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkaa1232

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.