TRF1, a duplex telomeric DNA binding protein, is implicated in homologous-recombination-based alternative lengthening of telomeres, known as ALT. However, how TRF1 promotes ALT activity has yet to be fully characterized. Here we report that Cdkdependent TRF1 phosphorylation on T371 acts as a switch to create a pool of TRF1, referred to as ( pT371)TRF1, which is recruited to ALT-associated PML bodies (APBs) in S and G2 phases independently of its binding to telomeric DNA. We find that phosphorylation of T371 is essential for APB formation and C-circle production, both of which are hallmarks of ALT. We show that the interaction of ( pT371)TRF1 with APBs is dependent upon ATM and homologous-recombination-promoting factors Mre11 and BRCA1. In addition, ( pT371)TRF1 interaction with APBs is sensitive to transcription inhibition, which also reduces DNA damage at telomeres. Furthermore, overexpression of RNaseH1 impairs ( pT371)TRF1 recruitment to APBs in the presence of campothecin, an inhibitor that prevents topoisomerase I from resolving RNA-DNA hybrids. These results suggest that transcription-associated DNA damage, perhaps arising from processing RNA-DNA hybrids at telomeres, triggers ( pT371)TRF1 recruitment to APBs to facilitate ALT activity.

Elevated replication stress is evident at telomeres of about 10-15% of cancer cells, which maintain their telomeres via a homologous recombination (HR)-based mechanism, referred to as alternative lengthening of telomeres (ALT). How ALT cells resolve replication stress to support their growth remains incompletely characterized. Here, we report that CSB (also known as ERCC6) promotes recruitment of HR repair proteins (MRN, BRCA1, BLM and RPA32) and POLD3 to ALT telomeres, a process that requires the ATPase activity of CSB and is controlled by ATM-and CDK2-dependent phosphorylation. Loss of CSB stimulates telomeric recruitment of MUS81 and SLX4, components of the structure-specific MUS81-EME1-SLX1-SLX4 (MUS-SLX) endonuclease complex, suggesting that CSB restricts MUS-SLX-mediated processing of stalled forks at ALT telomeres. Loss of CSB coupled with depletion of SMARCAL1, a chromatin remodeler implicated in catalyzing regression of stalled forks, synergistically promotes not only telomeric recruitment of MUS81 but also the formation of fragile telomeres, the latter of which is reported to arise from fork stalling. These results altogether suggest that CSB-mediated HR repair and SMARCAL1-mediated fork regression cooperate to prevent stalled forks from being processed into fragile telomeres in ALT cells.

TRF1, a component of the shelterin complex, plays a key role in both telomerase-dependent telomere maintenance and alternative lengthening of telomeres, the latter also known as ALT. Characteristics of ALT cells include C-circles and ALT-associated PML bodies, referred to as APBs. The function of TRF1 is tightly regulated by post-translational modification including phosphorylation, however TRF1 phosphorylation sites have yet to be fully characterized. Here we report a novel TRF1 phosphorylation site threonine 271. We show that a nonphosphorylatable mutation of T271A impairs TRF1 binding to telomeric DNA in vivo and renders TRF1 defective in inhibiting telomerase-dependent telomere elongation. On the other hand, TRF1 carrying a phosphomimic mutation of T271D is competent in not only binding to telomeric DNA but also inhibiting telomerase-mediated telomere lengthening. These results suggest that TRF1 phosphorylation on T271 negatively regulates telomerase-mediated telomere maintenance. We find that in telomerase-negative ALT cells, TRF1 carrying either a T271A or T271D mutation is able to promote C-circle production but fails to support APB formation. These results suggest that TRF1 phosphorylation on T271 is necessary for APB formation but dispensable for C-circle production. These results further imply that APB formation can be mechanistically separated from C-circle production.

Research conducted at McMaster University has shown a relative biological effect (RBE) of unity, between 280keV neutrons and 137Cs gamma radiation, for the induction of apoptosis in human lymphocytes. Similar results have also been seen by Vral et al. (1998) and Warenius and Down (1995) for the induction of apoptosis in human lymphocytes and mouse thymocytes respectively. It has previously been reported that neutrons of energies 36keV to 14.6Mev have an RBE ranging from 67.1±28.9 to 16±6.8 for induction of dicentrics in human lymphocytes respectively (Schmid et al., 2003, 2002, and 2000). This discrepancy between RBEs suggests that further work is needed to understand why human lymphocytes have such varying response to different radiations. Radiation‐induced apoptosis and chromosomal aberrations are being measured in parallel. In this study, it was found that 280keV neutron induce chromosome breaks with an RBE of 6.4 (for the induction of 1 break/cell). An experiment was established to study the types of chromosomal aberrations which exist in the lymphocytes which have not committed apoptosis 48 hours after radiation. Isolated lymphocytes were incubated for 48 hours in complete media before PHA stimulation. This allowed the analysis of the chromosomal damage present in cells that do not commit apoptosis immediately. It is possible that the cells which have not committed apoptosis at 48 hours are those which have been less severely damaged and may be viewed by the organism as being repairable.