RESUMO
The Fanconi anemia (FA) pathway repairs DNA interstrand crosslinks (ICLs) in humans. Activation of the pathway relies on loading of the FANCD2/FANCI complex onto chromosomes, where it is fully activated by subsequent monoubiquitination. However, the mechanism for loading the complex onto chromosomes remains unclear. Here, we identify 10 SQ/TQ phosphorylation sites on FANCD2, which are phosphorylated by ATR in response to ICLs. Using a range of biochemical assays complemented with live-cell imaging including super-resolution single-molecule tracking, we show that these phosphorylation events are critical for loading of the complex onto chromosomes and for its subsequent monoubiquitination. We uncover how the phosphorylation events are tightly regulated in cells and that mimicking their constant phosphorylation leads to an uncontrolled active state of FANCD2, which is loaded onto chromosomes in an unrestrained fashion. Taken together, we describe a mechanism where ATR triggers FANCD2/FANCI loading onto chromosomes.
Assuntos
Cromatina , Anemia de Fanconi , Humanos , Fosforilação , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Dano ao DNA , Ubiquitinação , Reparo do DNA , Proteínas Mutadas de Ataxia Telangiectasia/metabolismoRESUMO
The Fanconi anemia (FA) pathway repairs DNA interstrand crosslinks (ICLs). Many FA proteins are recruited to ICLs in a timely fashion so that coordinated repair can occur. However, the mechanism of this process is poorly understood. Here, we report the purification of a FANCD2-containing protein complex with multiple subunits, including WRNIP1. Using live-cell imaging, we show that WRNIP1 is recruited to ICLs quickly after their appearance, promoting repair. The observed recruitment facilitates subsequent recruitment of the FANCD2/FANCI complex. Depletion of WRNIP1 sensitizes cells to ICL-forming drugs. We find that ubiquitination of WRNIP1 and the activity of its UBZ domain are required to facilitate recruitment of FANCD2/FANCI and promote repair. Altogether, we describe a mechanism by which WRNIP1 is recruited rapidly to ICLs, resulting in chromatin loading of the FANCD2/FANCI complex in an unusual process entailing ubiquitination of WRNIP1 and the activity of its integral UBZ domain.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Reagentes de Ligações Cruzadas/química , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , ATPases Associadas a Diversas Atividades Celulares/química , Sequência de Aminoácidos , Sobrevivência Celular , Cromatina/metabolismo , Proteínas de Ligação a DNA/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Células HeLa , Humanos , Modelos Biológicos , Domínios Proteicos , Subunidades Proteicas/metabolismo , UbiquitinaçãoRESUMO
Interstrand crosslinks (ICLs) of the DNA helix are a deleterious form of DNA damage. ICLs can be repaired by the Fanconi anemia pathway. At the center of the pathway is the FANCD2/FANCI complex, recruitment of which to DNA is a critical step for repair. After recruitment, monoubiquitination of both FANCD2 and FANCI leads to their retention on chromatin, ensuring subsequent repair. However, regulation of recruitment is poorly understood. Here, we report a cluster of phosphosites on FANCD2 whose phosphorylation by CK2 inhibits both FANCD2 recruitment to ICLs and its monoubiquitination in vitro and in vivo. We have found that phosphorylated FANCD2 possesses reduced DNA binding activity, explaining the previous observations. Thus, we describe a regulatory mechanism operating as a molecular switch, where in the absence of DNA damage, the FANCD2/FANCI complex is prevented from loading onto DNA, effectively suppressing the FA pathway.
