Abstract
Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes from incomplete replication, degradation and detection as DNA breaks. Mammalian telomeres are protected by shelterin, a multiprotein complex that binds the TTAGGG telomeric repeats and recruits a series of additional factors that are essential for telomere function. Although many shelterin-associated proteins have been so far identified, the inventory of shelterin-interacting factors required for telomere maintenance is still largely incomplete. Here, we characterize AKTIP/Ft1 (human AKTIP and mouse Ft1 are orthologous), a novel mammalian shelterin-bound factor identified on the basis of its homology with the Drosophila telomere protein Pendolino. AKTIP/Ft1 shares homology with the E2 variant ubiquitin-conjugating (UEV) enzymes and has been previously implicated in the control of apoptosis and in vesicle trafficking. RNAi-mediated depletion of AKTIP results in formation of telomere dysfunction foci (TIFs). Consistent with these results, AKTIP interacts with telomeric DNA and binds the shelterin components TRF1 and TRF2 both in vivo and in vitro. Analysis of AKTIP- depleted human primary fibroblasts showed that they are defective in PCNA recruiting and arrest in the S phase due to the activation of the intra S checkpoint. Accordingly, AKTIP physically interacts with PCNA and the RPA70 DNA replication factor. Ft1-depleted p53-/- MEFs did not arrest in the S phase but displayed significant increases in multiple telomeric signals (MTS) and sister telomere associations (STAs), two hallmarks of defective telomere replication. In addition, we found an epistatic relation for MST formation between Ft1 and TRF1, which has been previously shown to be required for replication fork progression through telomeric DNA. Ch-IP experiments further suggested that in AKTIP-depleted cells undergoing the S phase, TRF1 is less tightly bound to telomeric DNA than in controls. Thus, our results collectively suggest that AKTIP/Ft1 works in concert with TRF1 to facilitate telomeric DNA replication.
Highlights
Mammalian telomeres consist of double stranded TTAGGG repeats that terminate with a single stranded 3' overhang
AKTIP-depleted chromosomes exhibit an accumulation of DNA repair factors at their ends, which are diagnostic of telomere damage
Our results indicate that AKTIP cooperates with the shelterin component TRF1 to ensure proper telomere replication
Summary
Mammalian telomeres consist of double stranded TTAGGG repeats that terminate with a single stranded 3' overhang. These repeats are added to chromosome ends by telomerase to compensate for the terminal DNA loss that occurs at each replication cycle due to the intrinsic inability of the DNA replication machinery to duplicate chromosome ends [1]. The TTAGGG repeats bind a telomere-specific six-subunit protein complex, called shelterin, that inhibits the DNA damage response (DDR) at chromosome ends and regulates telomerase activity [2]. Deletion of TRF2 activates ATM signaling and the Non Homologous End Joining (NHEJ) DNA repair pathway leading to telomeric fusions (TFs). Loss of TRF1 activates ATR/ ATM signaling and disrupts telomere replication [6, 7]
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