Telomeres are very challenging genomic regions for the replication complex progress due to the their specific features; indeed the presence of heterochromatin, of secondary structures such as G-quadruplex and T-loop, TERRA transcription, as well as telomere compaction and telomere attachment to the nuclear envelope can be sources of endogenous blocks for replication forks. The shelterin TRF1 plays a key role in ensuring proper telomere replication; in its absence were observed the formation of telomeric aberration linked to replication impairment, as fragile telomeres. It likely acts mainly by recruiting specific factors to telomeres in S-phase, such as helicases BLM and RTEL1, that unwind telomeric secondary structure to allow replication fork complex passage. However the precise mechanism of action is still elusive and probably other factors remain to be discovered.
AKTIP is a telomeric protein that interacts with shelterins TRF2 and TRF1 and its important for proper telomere replication. Indeed its absence triggers the formation of aberrant telomeres, in particular fragile telomeres and sister telomere association, and affects the incorporation of BrdU at telomeres, both indicative of replication defects. This replication impairment induces the DNA damage response activation culminating in senescence induction in primary cells. AKTIP also interacts with replisome components RPA70 and PCNA. Even though AKTIP is clearly involved in telomere replication, its mechanism of action remains elusive. The aim of this project is investigate how AKTIP acts on telomere replication moving from the hypothesis that AKTIP forms a complex with shelterin TRF1 at telomeres in S-phase that contributes to correct recruitment of DNA helicases, BLM, WRN and RTEL1, at telomeres and to fork complex stabilization at these genomic regions. This study could also give insight in the definition of molecular mechanism of telomere replication, which is essential for genome integrity maintenance
Telomeres are very challenging genomic region for the replication complex progress due to the presence of heterochromatin, G-rich regions that are prone to forming secondary structures such as G-quadruplex and T-loop, telomere repeat containing RNA (TERRA) transcription including R-loop and RNA:DNA hybrids, as well as telomere compaction and telomere attachment to the nuclear envelope. All these features can be sources of endogenous blocks that impede the progression of the fork through telomeric tracts (3). A key role in ensuring proper telomere replication is managed by shelterins that recruits factor enabling to solve the topological and structural matters that are present at the end of chromosomes. In particular TRF1 promotes telomere replication and its absence induces replication fork stall at telomeres triggering the formation fragile telomeres. It likely acts recruiting BLM helicase to unwind G-quadruplex and also several other proteins including RTEL1, TopIIalpha and Timeless, but its precise mechanism of action is still elusive and probably other factors remain to be discovered (3,6-8).
AKTIP is a shelterin interacting protein that as a role in telomere and general DNA replication (9,11). The principal aim of this project is to deepen the role of AKTIP in telomere metabolism. Our hypothesis is that AKTIP could form a complex with shelterin TRF1 at telomeres in S-phase and that this complex could contribute to correct recruitment of DNA helicases, BLM, WRN and RTEL1, at telomeres and to replication fork complex stabilization at these genomic regions. In this way AKTIP contributes to faithful telomeres replication.
Beyond elucidating the role of AKTIP at telomeres the proposed project could also give a contribution on the understanding of the molecular mechanisms of telomere replication, that is not completely explained, but that it's a fundamental process to ensure proper genome maintenance and avoiding DNA damage and senescence. Moreover, recently, telomeric replication stress was also described as a driver for accelerated tumorigenesis (12) underlying the importance of a better comprehension of this process.
The proposed project is based on robust preliminary results both already published and unpublished. It is also organized in subtasks which are designed as a logical sequence, planned to give results independently, and, at the same time, to produce an integrative picture of the role of AKTIP at telomere replication in complex with the shelterin TRF1. All these elements should ensure a positive success/risk ratio for this project.
Depending on the accorded grant I will develop one or more subtasks.