Defining RAD54 function in the alternative lengthening of telomeres pathway
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BACKGROUND: Telomeres are the DNA sequences at the end of chromosomes that are made up of highly repetitive sequences to protect the ends of chromosomes from damage. Telomeres shorten with each round of DNA replication eventually causing cellular senescence. Many cancer cells are able to overcome or evade senescence by elongating their telomeres. Alternative lengthening of telomeres (ALT) is a recombination based mechanism used by cancer cells to maintain telomere length. Evidence supports that unresolved replication stress at telomeric DNA promotes telomere elongation. Given the role of RAD54 in recombination genome wide, we were interested in investigating whether RAD54 is contributing to ALT telomere maintenance. OBJECTIVE: To define the role of RAD54 in ALT telomere maintenance. METHODS: Several different known ALT cell lines and non-ALT cell lines were examined using wet-lab techniques. Combined immunofluorescence and DNA FISH (IF-FISH) was used to visualize co-localization between RAD54 and telomeres, siRNA was used to deplete specific mRNA, and western blots were used to confirm these knockdowns. RESULTS: IF-FISH showed enrichment of RAD54 at the telomeres in ALT cells as compared to non-ALT cells. There was a decrease in incorporation of the synthetic nucleotide EdU in the absence of RAD54, indicating a decrease in DNA synthesis. No change was seen in the recruitment of RAD51, a recombinase, to telomeres in the absence of RAD54. There was a significant increase in MUS81 colocalization to ALT telomeres in the absence of RAD54 and an increase in the number of ultra-fine anaphase bridges. CONCLUSIONS: Using combined immunofluorescence and DNA FISH, we found enrichment of RAD54 at the telomeres in ALT cells as compared to non-ALT cell lines. Furthermore, RAD54 is predominantly found at ALT telomeres in ALT-associated promyelocytic leukemia (PML) bodies (APBs), which are nuclear condensates containing telomeres and many repair proteins. APBs are thought to be sites of active recombination, mediated by the recombinase RAD51. We can monitor recombination using EdU incorporation events at telomeres. RAD54 promotes DNA synthesis events at ALT telomeres, as measured through EdU incorporation. No change was found in RAD51 recruitment to telomeres in the absence of RAD54, indicating that RAD54 is not required for RAD51 mediated synapsis. By over-expressing RAD54 mutants, we found that sites of DNA synthesis, that are thought to be elongation events at ALT telomeres, are dependent on the ATPase and branch migration activities of RAD54. These results suggest that RAD54 is promoting telomere elongation by mediating the migration of the branched DNA structures formed at telomeres during recombination. When RAD54 is depleted, we found an increase in recruitment of the nuclease MUS81 to ALT telomeres, suggesting that in the absence of branch migration, ALT telomeres are cleaved to resolve recombination intermediates. Together, these data demonstrate a crucial role for RAD54 in promoting ALT mediated telomere elongation through resolving homologous recombination intermediates via branch migration.