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

Abstract

Summary StatementTRF2 is recruited to damage sites by a two-step mechanism and functions in non-sister chromatid homologous recombination repair Abstract TRF2 is a shelterin component critical for telomere integrity. While TRF2 directly recognizes and binds telomeric repeats, evidence suggests that it also localizes to nontelomeric 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(ADP-ribose) polymerase (PARP)-dependent and requires positively charged amino acid residues in the Nterminal domain. The phase II recruitment is through the C-terminal MYB/SANT domain and requires the iDDR region in the hinge domain of TRF2. Phase II is mediated by the MRE11 complex and is stimulated by hTERT. PARP-dependent recruitment of intrinsically disordered proteins contributes to transient displacement of TRF2 that separates phases I and II. TRF2 depletion specifically affects non-sister chromatid homologous recombination (HR) repair, but not HR between sister chromatids or classic and alternative non-homologous endjoining. Our results demonstrate a unique recruitment mechanism and function of TRF2 at non-telomeric DNA damage sites.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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 ( Figure 6 A and Figure 7 A) and TRF2 ( Figure 7 A and Figure 8 A) are typically dispersed across the nucleoplasm whereas LAP1 is mostly distributed throughout the NE ( Figure 6 A and Figure 8 A), 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 ( Figure 2 C, Figure 4 B and Figure 5 B).…”

“…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 ].…”

Laser Scissors and Tweezers to Study Chromosomes: A Review

“…Notwithstanding this variability, it has become clear that laser microirradiation of the DNA/chromatin have contributed significantly to understanding the DNA damage recognition and repair processes. These studies have involved laser damage and repair in the interphase nucleus ( Truong et al, 2013 ; Wang et al, 2013 , 2014 ; Kim et al, 2015 ; Saquilabon Cruz et al, 2016 ; Kong et al, 2018 ; Murata et al, 2019 ), and on condensed chromosomes in mitosis ( Gomez-Godinez et al, 2010 ; Silva et al, 2013 ).…”