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Specific mutations in the replacement histone variant H3.3 are known to drive cancers such as glioblastomas, chondroblastomas and large cell tumors of the bone primarily in children and young adults. Several recent studies have suggested that transcriptional defects associated with H3.3 mutations are likely to be involved in tumor formation. However, transcription independent roles of H3.3 and their potential contribution to cancer have not been investigated. Here we report that histone H3.3 (but not the related replication dependent H3.1 or H3.2 variants) is recruited within ~60 seconds to sites of laser induced DNA damage. This recruitment is dependent on acetylation at the N terminus of the protein. A stable depletion of this protein in human cells leads to high levels of endogenous DNA damage and an impaired ability to repair this damage, leading to genomic instability and sensitivity to DNA damaging agents. Flies with reduced levels of H3.3 are also sensitive to damaging agents and accumulate spontaneous damage. H3.3 depletion alters the recruitment of double strand break (DSB) repair factors, with defects in the recruitment of proteins involved in homologous recombination (HR) repair and faster recruitment of some proteins involved in non-homologous end joining (NHEJ). We also find that the cancer associated mutants are defective in localizing to damage sites and their expression leads to an accumulation of high levels of endogenous damage. Overall, our data from evolutionarily distant species suggest that histone H3.3 plays a crucial role in HR-mediated DNA repair which is conserved across eukaryotes. Hence, based on the strong links between defective DNA repair and cancer, we propose that the DNA repair defects associated with H3.3 mutations are likely to contribute to genomic instability, and thereby to carcinogenesis, independent of the known transcriptional roles of histone H3.3. Based on these findings, we propose a potential therapeutic strategy that combines the use of inhibitors along with a DSB inducing agent to selectively target cells that are defective in HR repair due to H3.3 depletion or mutation. Our promising preliminary data show that this is effective in H3.3 knockdown cells and may also be useful in killing H3.3 cancer mutants. This knowledge helps us to understand the complex details of these deadly mutations and hopefully brings us closer to a treatment.