Apollo at the nexus of DNA repair, telomere protection, and aging. – APOthesis

Aging is a complex process that involves cellular and organismal pathways and deciphering its determinants has become an important topic in modern science. Research on aging in Biology goes through the study of diseases characterized by premature aging, analysing physiological aging in multiple species and determining the molecular actors and mechanisms involved. Among these actors are telomeres, the natural ends of chromosomes that provide protection against the DNA damage response (DDR) and repair and ensure an efficient replication of these ends. In humans, germline mutations in factors involved in telomere protection cause, among other progeroid pathologies, the premature aging disorder Høyeraal-Hreidarsson syndrome (HH), a severe form of dyskeratosis congenita (DC), characterized by early onset bone marrow failure, intrauterine growth retardation, microcephaly, and immunodeficiency. Apollo/SNM1B (a 5' exonuclease belonging to the beta-CASP family) is a protein that is involved in the repair of both mitomycin C (MMC)-induced DNA interstrand crosslinks (ICL) and DNA double-strand breaks. Apollo also participates in the stabilization of stalled replication forks and S-phase checkpoint activation. Besides its function as a DNA repair factor genome-wide, Apollo was found by Partner 3 to be recruited to telomeres via its interaction with the telomeric protein TRF2 where it is involved in protecting telomeres and helping their replication. Apollo and TRF2 interact through a region of the C-terminal part of Apollo (TRFH Binding Motif, TBM) and the dimerization domain of TRF2 (TRFH). Phylogenetic analyses conducted by Partner 2 suggested that Apollo TBM domain has coemerged with TRF1/TRF2 to recognize the vertebrate-specific TRFH domain of TRF2. Thus, although Apollo appears to be an important factor necessary for telomere stability and global genome integrity, its relative contribution to telomere maintenance, general DNA repair and control of aging remains elusive. Interestingly, a recent comparative genomic analysis revealed the existence of sequence variations in the TBM domain of Apollo in long-lived Galapagos giant tortoises compared to short-lived turtles suggesting that the strength of the Apollo-TRF2 interaction might be a determinant of organismal lifespan.
We propose in APOthesis to investigate whether the telomeric and/or extra-telomeric functions of Apollo and the variations in sequences that could affect the strength of TRF2-Apollo interaction found in different species could contribute to cellular/organismal lifespan and aging phenotype. Strikingly, Partner 1 recently identified biallelic Apollo mutations in 3 unrelated individuals with HH. Interestingly, all three patients carry a homozygous or compound heterozygous (in combination with a null-allele) missense variant affecting the amino-acid L142 (i.e. L142F and L142S) located in the beta-lactamase domain of Apollo. Strikingly, coimmunoprecipitation experiments indicate that, although at distance from the TBM, these missense mutations reduce the interaction of Apollo with TRF2. Hence, in one hand we identified mutations in Apollo affecting TRF2-Apollo interaction and causing the progeroïd HH syndrome and, in the other hand, variations in the TRF2 binding site of Apollo correlate with a longer longevity in chelonians.
Based on these findings we hypothesized that Apollo, via, at least in part, its interaction with TRF2 could be a determinant of cellular and organismal lifespan.

We will use different models (Apollo-mutated cells from patients (available), human Apollo KO cell lines generated by CRISPR/Cas9 (available), Apollo and/or TRF2 mutated human cell lines generated by CRISPR/Cas9 (to produce), and Apollo L142S knock-in mouse model (available), to decipher the functions of Apollo at telomere, elsewhere in the genome, and determine their contributions in global cellular/organismal lifespan.