RESUMEN
CTCF is a zinc finger protein associated with transcription regulation that also acts as a barrier factor for topologically associated domains (TADs) generated by cohesin via loop extrusion. These processes require different properties of CTCF-DNA interaction, and it is still unclear how CTCF's structural features may modulate its diverse roles. Here, we employ single-molecule imaging to study both full-length CTCF and truncation mutants. We show that CTCF enriches at CTCF binding sites (CBSs), displaying a longer lifetime than observed previously. We demonstrate that the zinc finger domains mediate CTCF clustering and that clustering enables RNA recruitment, possibly creating a scaffold for interaction with RNA-binding proteins like cohesin's subunit SA. We further reveal a direct recruitment and an increase of SA residence time by CTCF bound at CBSs, suggesting that CTCF-SA interactions are crucial for cohesin stability on chromatin at TAD borders. Furthermore, we establish a single-molecule T7 transcription assay and show that although a transcribing polymerase can remove CTCF from CBSs, transcription is impaired. Our study shows that context-dependent nucleic acid binding determines the multifaceted CTCF roles in genome organization and transcription regulation.
Asunto(s)
Factor de Unión a CCCTC , Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , Cohesinas , ARN , Imagen Individual de Molécula , Dedos de Zinc , Humanos , Sitios de Unión , Factor de Unión a CCCTC/metabolismo , Factor de Unión a CCCTC/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , ADN/metabolismo , ADN/genética , Unión Proteica , ARN/metabolismo , ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Imagen Individual de Molécula/métodos , Transcripción GenéticaRESUMEN
SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.
