CRL4 ubiquitin ligase stimulates Fanconi anemia pathway-induced single-stranded DNA-RPA signaling.

ABSTRACT: BACKGROUND:DNA-crosslinking agents like cisplatin and mitomycin C (MMC) are indispensible for the treatment of many solid malignancies. These anticancer drugs generate DNA interstrand crosslinks (ICLs) that cause cell death by blocking replication forks. Many factors counteracting ICL-induced DNA replication stress, including the Fanconi anemia (FA) pathway, are regulated by ubiquitination and, therefore, ubiquitin ligases are potential targets for the sensitization of cancer cells to crosslinking agents. In this study, we investigated the function of the CRL4 ubiquitin ligase in modulating the response of cancer cells to ICL induction. METHODS:The two cullin paralogs CUL4A and CUL4B, which form the CRL4 ligase scaffold, were depleted in cancer cells by small interfering RNA followed by analysis of the cellular and biochemical responses to ICLs elicited upon cisplatin or MMC treatment. RESULTS:We report that the combined depletion of CUL4A and CUL4B weakens an FA pathway-dependent S phase checkpoint response. CRL4 positively stimulates the monoubiquitination of FANCD2 required for the recruitment of XPF-ERCC1, a structure-specific endonuclease that, in turn, contributes to the display of single-stranded DNA (ssDNA) at ICLs. After CRL4 down regulation, the missing ssDNA results in reduced recruitment of RPA, thereby dampening activation of ATR and CHK1 checkpoint kinases and allowing for S phase progression despite ICL induction. CONCLUSION:Our findings indicate that CRL4 promotes cell survival by potentiating an FA pathway-dependent ssDNA-RPA signaling platform installed at ICLs. The anticancer efficacy of crosslinking agents may, therefore, be enhanced by down regulating CRL4 activity.

SUBMITTER: Codilupi T 

PROVIDER: S-EPMC6833152 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature