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.