Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

Abstract

TRF2 (TERF2) binds to telomeric repeats and is critical for telomere integrity. Evidence suggests that it also localizes to non-telomeric DNA damage sites. However, this recruitment appears to be precarious and functionally controversial. We find that TRF2 recruitment to damage sites occurs by a two-step mechanism: the initial rapid recruitment (phase I), and stable and prolonged association with damage sites ( phase II). Phase I is poly(ADPribose) polymerase (PARP)-dependent and requires the N-terminal basic domain. The phase II recruitment requires the C-terminal MYB/ SANT domain and the iDDR region in the hinge domain, which is mediated by the MRE11 complex and is stimulated by TERT. PARPdependent recruitment of intrinsically disordered proteins contributes to transient displacement of TRF2 that separates two phases. TRF2 binds to I-PpoI-induced DNA double-strand break sites, which is enhanced by the presence of complex damage and is dependent on PARP and the MRE11 complex. TRF2 depletion affects non-sister chromatid homologous recombination repair, but not homologous recombination between sister chromatids or non-homologous endjoining pathways. Our results demonstrate a unique recruitment mechanism and function of TRF2 at non-telomeric DNA damage sites.

Nuclear Accumulation of LAP1:TRF2 Complex during DNA Damage Response Uncovers a Novel Role for LAP1

“…For this purpose, immunocytochemistry assays were performed to investigate the co-localization of endogenous LAP1, TRF2 and γ-H2AX in HeLa cells' nuclei in a baseline state and upon a short-term bleomycin exposure (200 µg/mL for 30 min, followed by 6 h of recovery). A general analysis by confocal microscopy revealed that all proteins are located at the expected nuclear compartments irrespective of the experimental condition, wherein γ-H2AX ( Figures 6A and 7A) and TRF2 ( Figures 7A and 8A) are typically dispersed across the nucleoplasm whereas LAP1 is mostly distributed throughout the NE (Figures 6A and 8A), being consistent with previous descriptions for each protein [18,22,44,59,60]. Regarding their fluorescence intensity in the nucleus, the results indicated that, overall, the abundance of the three proteins augments after cell exposure to the DNA-damaging treatment comparatively to the control group (Supplementary Figure S4A,C,E), thus confirming the prior findings ( Figures 2C, 4B and 5B).…”

“…After showing that a protein complex formed by LAP1 and TRF2 occurs in vitro, we investigated the establishment of this interaction in human cells. We hypothesized that it could be constitutively active in the cell or, instead, be triggered by a specific signaling event, more precisely by DDR activation, given the previously reported inhibitory [33][34][35][36][37][38][39] and stimulatory [40][41][42][43][44][45][46] roles of TRF2 as well as the recently suggested involvement of LAP1 [29] in this biological process. Before directly addressing this question, cellular models of DNA damage induction were developed; these consisted in exposing HeLa cells to different concentrations of H 2 O 2 -a reactive oxygen species (ROS)-or bleomycin-a radiomimetic anticancer drug-for distinct time periods.…”

“…In sharp contrast with this inhibitory action on DDR mechanisms, several lines of evidence indicate that TRF2 is additionally directly involved in DSB repair events occurring in extra-telomeric regions. In short, TRF2 is rapidly and transiently recruited to DNA damage sites located outside of telomeres at a very initial stage of genomic DNA break recognition [40][41][42][43][44]. The efficiency of TRF2 clustering at non-telomeric DNA increases in response to high doses of DSBs with complex lesions (e.g., crosslinking and base damage) [43], suggesting that TRF2 may act as an early sensor of this type of DNA damage and/or as a mediator of its effective resolution.…”

“…The efficiency of TRF2 clustering at non-telomeric DNA increases in response to high doses of DSBs with complex lesions (e.g., crosslinking and base damage) [43], suggesting that TRF2 may act as an early sensor of this type of DNA damage and/or as a mediator of its effective resolution. Once at the injured chromatin regions, TRF2 is essential for directing the processing of genomic DSBs towards a more accurate repair pathway of HR, likely through its ability to stimulate intra-chromatid strand invasion [44,45], though a possible role in the fast, error-prone repair pathway of NHEJ has also been suggested [46]. Importantly, the only previous report of an association between LAP1 and TRF2 (and with RAP1 as well) came from a genome-wide study dedicated to the identification of candidate human proteins that bind to the six core shelterin complex subunits [47].…”

Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

“…By binding to the viral TMR, TRF2 may play a similar role by shielding the end of the viral genome from DDR. Second, TRF2 has been shown to participate in HR [70,71]. We therefore surmise that the presence of TRF2 at viral DNA favors chromosomal integration by facilitating HR events between the host telomeres and viral telomeric sequences.…”