Abstract
RAD51 T131P–expressing cells display defective ICL repair but normal HR-mediated repair.
Major finding: RAD51 T131P–expressing cells display defective ICL repair but normal HR-mediated repair.
Mechanism: RAD51 T131P has constitutive ATPase activity and impaired DNA-pairing and strand-exchange functions.
Impact: RAD51 protects DNA and maintains genomic integrity during ICL repair independent of its function in HR.
The Fanconi anemia (FA) pathway orchestrates DNA interstrand crosslink (ICL) repair, a multistep process that involves DNA incision, translesion synthesis, and homologous recombination (HR). Mutational inactivation of FA genes leads to developmental defects, cancer predisposition, bone marrow failure, and hypersensitivity to crosslinking agents. Recent work suggests that the HR recombinase RAD51 may be recruited to stalled replication forks prior to FA pathway activation. Consistent with this idea, Wang and colleagues identified a heterozygous mutation in RAD51 (c.391A>C) in a patient with FA symptoms, which resulted in an amino acid change (T131P) at a residue required for ATP binding and hydrolysis. Patient-derived RAD51-mutant cells exhibited hypersensitivity to DNA-crosslinking agents, which was reversed upon overexpression of wild-type RAD51 or CRISPR-mediated knockout of the mutant allele, and defective DNA repair resolution despite an activated FA pathway. Surprisingly, although the RAD51 T131P protein was unable to localize to chromatin and delayed the formation of wild-type RAD51 foci, cells expressing RAD51 T131P were HR proficient. Expression of RAD51 T131P augmented the phosphorylation of the single-stranded DNA (ssDNA) binding protein replication protein A in response to ICL-inducing agents; ssDNA accumulation in RAD51 T131P–expressing cells was attributed to increased activity of the resecting nucleases DNA2 and WRN. Moreover, RAD51 T131P was characterized by unregulated DNA-independent ATPase activity and an inability to promote DNA pairing and strand exchange. Co-mixing of different ratios of mutant and wild-type RAD51 in vitro revealed that both the DNA-pairing and strand-exchange activities of wild-type RAD51 were inhibited by mutant RAD51. However, the dominant behavior observed in vitro was seen only when mutant RAD51 was more abundant than the wild-type protein, explaining why patient cells, in which only 20% of RAD51 is mutant, are HR proficient. These findings show the role of RAD51 in protection of DNA during ICL repair independent of its function in HR and highlight an additional clinical subclass of Fanconi anemia.
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