Tuesday, September 26, 2023
9/26/2023
PROJECT SUMARY Cancer cells ensure replicative immortality through activation of telomere maintenance mechanisms. Approximately 15% of cancers indefinitely lengthen their telomeres through a homology directed repair (HDR) driven mechanism, termed Alternative Lengthening of Telomeres (ALT). Telomere lengthening via the ALT mechanism is reliant upon two non-canonical HDR pathways: (1) RAD51-HOP2 dependent homologous recombination and (2) RAD52 mediated de novo telomere synthesis via the PCNA-RFC1-Pold replisome. Our lab has previously shown that RAD51AP1, an accessory HR protein, is an essential mediator of both ALT-HDR pathways. Depletion of RAD51AP1 results in telomere shortening of ALT positive (ALT+) cancer cells. However, the exact mechanism behind RAD51AP1 mediated telomere lengthening via ALT-HDR remains unknown. Interestingly, our preliminary data show that disruption of RAD51AP1 results in a decrease of telomere repeat- containing lincRNA (TERRA). Additionally, our preliminary data identify an ALT specific RAD51AP1 interactome containing multiple proteins involved in HDR-coupled transcriptional silencing. Dysregulation of TERRA has been linked to insufficient telomere HDR. Therefore, understanding the processes and key proteins involved in TERRA mediation within ALT cancers will provide essential information toward the development of ALT therapeutics. I hypothesize that RAD51AP1 stabilizes TERRA RNA-DNA hybrids at ALT telomeres and serves as a hub for the assembly of the critical mediators of ALT-HDR. I will investigate the role of RAD51AP1 in the regulation of TERRA in ALT through two aims. Aim 1 will characterize the interaction between RAD51AP1 and TERRA. The proposed experiments will identify the RNA interacting region of RAD51AP1 and determine the regulatory effect of RAD51AP1 on telomeric TERRA RNA-DNA hybrid stability through a series of co-immunoprecipitation and immunofluorescence experiments. This aim will establish a novel characteristic of RAD51AP1 in its interaction with TERRA. Aim 2 will define the role of functional interplay between RAD51AP1 and TERRA during ALT-HDR. The proposed experiments will identify the sequential mechanism of RAD51AP1 regulation of TERRA by visualizing the epistatic effects of depletion of TERRA, RAD51AP1, and the RNA regulating proteins from the RAD51AP1 interactome. Additionally, the mechanism of action in which RAD51AP1 regulates TERRA will be deciphered through observation of how the depletion of RAD51AP1 and RNA regulating RAD51AP1-interactors effect transcription coupled HDR mechanisms in ALT cells. This aim will elucidate the function of RAD51AP1 at the intersection of DNA-RNA mediated HDR in ALT. Completion of these aims will provide insight into essential regulatory mechanisms within the ALT pathway and potential protein targets that will advance the development of ALT therapies.
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