Abstract
TRF2 is part of the shelterin complex that hides telomeric DNA ends and prevents the activation of the cNHEJ pathway that can lead to chromosomal fusion. TRF2, however, also actively suppresses the cNHEJ pathway by recruiting two proteins, MRE11 and UBR5. MRE11 binds BRCC3, which in turn deubiquitinates γH2AX deposited at exposed telomeric DNA ends and limits RNF168 recruitment to the telomere. UBR5, in contrast directly ubiquitinates and destroys RNF168. The loss of telomeric RNF168 in turn blocks the subsequent recruitment of 53BP1 and prevents the cNHEJ-mediated fusion of chromosomes with exposed telomeric DNA ends. Although MRE11 and UBR5 are both involved in the control of telomeric RNF168 levels and the chromosome fusion process, their relative contributions have not been directly addressed. To do so we genetically suppressed MRE11 and UBR5 alone or in combination in glioma cell lines which we previously showed contained dysfunctional telomeres that were dependent on TRF2 for suppression of telomeric fusion and monitored the effects on events associated with telomere fusion. We here show that while suppression of either MRE11 or UBR5 alone had minimal effects on RNF168 telomeric accumulation, 53BP1 recruitment, and telomeric fusion, their combined suppression led to significant increases in RNF168 and 53BP1 telomeric recruitment and telomeric fusion and eventually cell death, all of which were reversible by suppression of RNF168 itself. These results show that MRE11 and UBR5 co-operate to suppress fusion at dysfunctional telomeres.
Highlights
Human telomeres are DNA-protein complexes present at the end of chromosomes
We previously showed that siRNA-mediated suppression of MRE11 did not alter telomeric levels of RNF168, 53BP1, or telomeric fusion in alternative lengthening of telomeres (ALT)-dependent (MGG119) or ALTindependent (MGG152) cells when assessed at 24h post suppression [12]
Introduction of constructs encoding siRNA-resistant forms of MRE11 and UBR5 in MGG119 cells with suppressed levels of these targets (Figures 1I–K), reversed the increase in the number of RNF168 foci per DAPI-stained nucleus. These results show that suppression of MRE11 or UBR5 alone is insufficient to drive the process that leads to telomeric fusion
Summary
Human telomeres are DNA-protein complexes present at the end of chromosomes. The DNA component of telomeres consists of double stranded 5’-TTAGGG-3’ DNA repeats and a single stranded G-rich 3’ overhang [1]. The DNA repeats fold back on themselves to form a T-loop structure while the 3’overhang inserts into the duplex repeats to form a D-loop that hides the terminal DNA sequence [2]. Both T- and D-loops are stabilized by the protein component of the telomere known as the shelterin cap. The shelterin cap is a 6-protein structure that interacts with telomeric DNA, prevents exposure of DNA ends, and limits DNA repair pathways that would otherwise lead to telomeric DNA fusion, genomic instability, and cell death [3, 4].
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