Abstract
Our genome is under constant threat from DNA damage that inflicts different kinds of lesions including DNA double-strand breaks (DSBs). Failure to correctly repair DSBs can cause gross chromosomal aberrations, which are a hallmark of cancer. Cells have evolved two major pathways to repair DSBs: non-homologous end-joining (NHEJ) and homologous recombination (HR). Human CtIP promotes DNA-end resection, which commits cells to error-free HR and prevents aberrant repair by NHEJ, hence it is a critical determinant of DSB repair pathway choice. However, the cellular response to DNA damage involves a complex interplay between different genome maintenance pathways and the role of CtIP in this multifaceted network is still poorly understood.
In the first part of my PhD study, we addressed the functional interplay between CtIP- dependent resection and the Fanconi anemia (FA) pathway during the repair of DNA interstrand crosslinks (ICLs). Fanconi anemia is an inherited disorder associated with a high risk to develop cancer and is caused by mutations in sixteen FA genes. Together, the FA proteins orchestrate ICL incision, translesion synthesis and HR. We demonstrate that chromatin association of CtIP in response to ICL-induced damage is strictly dependent on a functional FA core complex and FANCD2 monoubiquitination. Furthermore, we show that CtIP recruitment to ICL lesions is mediated by its direct interaction with FANCD2 and might be further reinforced by the discovered ability of CtIP to recognize ubiquitinated substrates. Remarkably, cells lacking FANCD2 akin to CtIP-depleted cells are impaired in DNA-end resection. We have identified FANCD2-binding sites on CtIP and provide evidence that CtIP-FANCD2 complex is required for the faithful repair of ICLs, meanwhile counteracting mutagenic NHEJ pathway. Interestingly, the phenotypes of FA cells such as genome instability and ICL hypersensitivity are further aggravated by CtIP depletion, indicating the significance of CtIP even in the absence of proficient FA pathway. Taken together, our data establish FANCD2 as a critical regulator of CtIP- mediated DNA-end resection and emphasize the essential role of CtIP in maintaining genome stability in response to ICL damage.
In the second part, we examined the phenotypes of the separation-of-function (S) mutations in human RAD50, a subunit of the MRE11-RAD50-NBS1 (MRN) complex that interacts with CtIP and plays crucial roles in DSB signaling and processing. We demonstrate that RAD50S mutants compromise resection and repair of DSBs induced specifically by DNA topoisomerase poisons, but do neither alter MRN complex integrity nor significantly affect MRN-dependent signaling in response to other types of DNA damaging agents. Based on our biochemical data we suggest that these phenotypes are caused by the impaired interaction between RAD50S mutants and CtIP, which is particularly important for the processing of topoisomerases trapped to DNA ends. Collectively, our results establish a key role for CtIP in the repair of ICLs and also highlight the significance of CtIP-MRN association for the processing of toxic protein- DNA adducts. Work presented in my thesis thus advances the understanding of how the DNA-end resection activity of CtIP is regulated to preserve genome stability.
Murina, Olga