Physical and Functional Association of the MRN Complex with Human Telomerase in Multiple Myeloma.

Telomerase is a specialized RNA-directed DNA polymerase that extends telomeres of eukaryotic chromosomes, is repressed in normal human somatic cells but is activated during development and upon malignant transformation. There is abundant evidence that the regulation of telomerase is multifactorial in mammalian cells, involving telomerase gene expression, post-translational protein-protein interactions, and protein phosphorylation. Initial experiments indicated that the catalytic subunit(s) of human telomerase (hTERT) is highly elevated in Multiple Myeloma (MM) cell lines and MM patient samples relative to normal donor plasma cells. We then exploited this highly detectable level of hTERT to address the potential role of proteins that may physically associate with the telomerase complex in MM. To this end, we investigated whether the MRN complex, a trimeric structure that consists of Mre11, RAD50 and Nbs that is evolutionarily conserved and functions in DNA repair and homologous recombination physically associates with the human telomerase complex. The MRN complex is required in vivo for a 5′ to 3′ exonuclease activity that mediates DNA recombination at double-strand breaks (DSBs). We have observed that each of the three MRN components was associated with the catalytic subunit of human telomerase (hTERT) in MM cell lines as demonstrated by in vivo co-immunoprecipitation with an hTERT monoclonal antibody under non-denaturing conditions. In addition, polyclonal antibodies to each MRN component individually immunoprecipitated the hTERT catalytic subunit. We also detected that the telomerase complex accessory proteins TRF-1 and TRF-2 are also associated with hTERT and the MRN components. TRF-2 may function as bridge coupling hTERT to the MRN complex. The association of the MRN components with hTERT was increased following treatment of myeloma cells with DNA-damaging agents indicating a functional relationship between telomere maintenance and the DNA repair pathway. The functional role of this complex is being addressed by measuring telomerase activity, telomere length and homologous recombination activity. Based upon the data that abolition of exonuclease activity in MRN mutants resulted in shortened telomeric DNA tracts, we hypothesize that this elevated MRN complex expression and its interaction with hTERT in myeloma, provides the ability to maintain telomeres and may be an important therapeutic target in MM.