RESUMEN
SLX4, a coordinator of multiple DNA structure-specific endonucleases, is important for several DNA repair pathways. Noncovalent interactions of SLX4 with ubiquitin are required for localizing SLX4 to DNA interstrand crosslinks (ICLs), yet how SLX4 is targeted to other functional contexts remains unclear. Here, we show that SLX4 binds SUMO-2/3 chains via SUMO-interacting motifs (SIMs). The SIMs of SLX4 are dispensable for ICL repair but important for processing CPT-induced replication intermediates, suppressing fragile site instability, and localizing SLX4 to ALT telomeres. The localization of SLX4 to laser-induced DNA damage also requires the SIMs, as well as DNA end resection, UBC9, and MDC1. Furthermore, the SUMO binding of SLX4 enhances its interaction with specific DNA-damage sensors or telomere-binding proteins, including RPA, MRE11-RAD50-NBS1, and TRF2. Thus, the interactions of SLX4 with SUMO and ubiquitin increase its affinity for factors recognizing different DNA lesions or telomeres, helping to direct the SLX4 complex in distinct functional contexts.
Asunto(s)
Genoma , Recombinasas/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Daño del ADN , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Recombinasas/genética , Alineación de Secuencia , Transducción de Señal , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Telómero/efectos de la radiación , Ubiquitina/genética , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitinas/genética , Rayos UltravioletaRESUMEN
The ATR (ATM [ataxia telangiectasia-mutated]- and Rad3-related) checkpoint is a crucial DNA damage signaling pathway. While the ATR pathway is known to transmit DNA damage signals through the ATR-Chk1 kinase cascade, whether post-translational modifications other than phosphorylation are important for this pathway remains largely unknown. Here, we show that protein SUMOylation plays a key role in the ATR pathway. ATRIP, the regulatory partner of ATR, is modified by SUMO2/3 at K234 and K289. An ATRIP mutant lacking the SUMOylation sites fails to localize to DNA damage and support ATR activation efficiently. Surprisingly, the ATRIP SUMOylation mutant is compromised in the interaction with a protein group, rather than a single protein, in the ATR pathway. Multiple ATRIP-interacting proteins, including ATR, RPA70, TopBP1, and the MRE11-RAD50-NBS1 complex, exhibit reduced binding to the ATRIP SUMOylation mutant in cells and display affinity for SUMO2 chains in vitro, suggesting that they bind not only ATRIP but also SUMO. Fusion of a SUMO2 chain to the ATRIP SUMOylation mutant enhances its interaction with the protein group and partially suppresses its localization and functional defects, revealing that ATRIP SUMOylation promotes ATR activation by providing a unique type of protein glue that boosts multiple protein interactions along the ATR pathway.