Rad18 Regulates BRCA2, FANCD2, and Rad51 to Repair Topoisomerase 1 Inhibitor‐Induced DNA Lesions through Homologous Recombination
Authors: Palle Komaraiah , Tripathi Kaushlendra , Clark David W , Mani Chinnadurai 
Published: November 2023

DNA topoisomerase 1 (Top1) is an essential enzyme in higher eukaryotes, which resolves topological barriers during most of the critical cellular processes involving DNA, including replication, transcription, recombination and repair. Top1 modulates DNA topology by introducing a transient single strand break through active site tyrosine(Tyr723) forming a covalent 3′‐phosphotyrosyl bond with the DNA, which is called Top1‐DNA covalent or cleavage complex (Top1cc). The torsional strain in the DNA drives the broken strand to rotate around the intact strand thereby relieving the extent of super coiling during the period between the cleavage and religation, allowing transcription or replication to proceed. In normal conditions, Top1ccis rapidly followed by a second transesterification reaction that reseals the broken strand to maintain the integrity of the DNA. The anticancer agent camptothecin(CPT) and its analogs (topotecan and irinotecan) prolong the stabilization of normally ephemeral complex of Top1 on nicked DNA (Top1cc). These CPT stabilized Top1cc are not known to be cytotoxic by themselves and are readily reversible after removal of the drug. However, prolonged stabilization of Top1cc creates multiple problems. Firstly, the failure to relieve super coiling generated by such processes as transcription and replication can lead to replication stress by creating torsional strain within the DNA. Furthermore, collision between an active replication fork and the Top1cc is capable of generating DNA double strand breaks (DSB) which can introduce mutations or lead to cell death. Although Top1 poisoning by CPT known to induce replication‐associated DSB, the mechanisms of their repair are poorly understood. Genetic screens in the model organism yeast and cancer cells have identified homologous recombination (HR)as the predominant DNA repair pathway involved in the repair of CPT‐induced DSB. Correspondingly, deficiencies in proteins that are involved in HR (ex. Rad51)as well as associated proteins such as Rad18, members of the Fanconi anemia(FA) family and the breast cancer associated (BRCA1, BRCA2) proteins have been shown to hypersensitize cells to CPT. We and others have previously shown that Rad18 regulates the FA pathway in response to fork stalling lesions induced by several agents, including Top1 poison CPT. Here our studies further demonstrate that in response to CPT, E3 ligase Rad18 is important for efficient FANCD2 monoubiquitination, and CPT‐induced FANCD2, BRCA2 and Rad51 nuclear foci formation and their colocalization. Furthermore, co‐immunoprecipitation studies revealed a physical as well as functional interaction between Rad18, FANCD2, BRCA2 and Rad51. Interestingly, our studies revealed reduced levels of BRCA2and Rad51 in the nucleus and their co‐localization in both Rad18‐ and FANCD2‐deficient cells in response to CPT. Moreover, loss of any or combination of these proteins also caused similar levels of HR deficiency, sensitization to CPT, and increased chromosomal aberrations (CA), suggesting these proteins play an important role in accurate repair of CPT‐induced, replication‐coupled DSB. Together our data show that Rad18 plays an important role in maintaining the epistatic relationship between multiple repair proteins to promote HR by regulating monoubiquitination of FANCD2, and thereby BRCA2 and Rad51 stability and repair foci in response to replication‐coupled DSB induced by CPT.Support or Funding InformationThis work is supported by NIH grant R01GM098956