| Popis: |
Telomeres are repetitive nucleoprotein complexes that protect the ends of linear chromosomes and prevent their detection as double strand breaks (DSBs), thereby averting activation of a DNA damage response (DDR). While these functions are clearly essential for maintaining genome integrity, it is intriguing to consider how DSBs within telomeres themselves are handled. In cycling cells, telomeric DSBs can be repaired by homologous recombination (HR) and alternative nonhomologous end-joining (ALT NHEJ). Localization of 53BP1, an important regulator of resection at broken ends, to damaged telomeres has also only been observed in replicating cells. Here, we characterized the cellular response to enzymatically induced telomeric DSBs, specifically in non-replicating G1 normal human fibroblasts and cancer cells. Telomeric DSBs in G1 human cells elicited early signatures of a DDR in that gamma (γ)-H2AX and MDC1 were recruited to broken telomeres. Consistent with previous reports, 53BP1 was not observed at telomeric break sites in G1. Furthermore, evidence of classical NHEJ (cNHEJ), the primary pathway of DSB repair in G1 mammalian cells, was lacking at broken telomeres. Likewise, no evidence of classical HR-dependent repair of telomeric DSBs in G1 was observed, as neither RAD51, RAD52, nor repair associated DNA synthesis were detected. Rather, and consistent with rapid truncation events and overall telomere shortening, telomeric DSBs in G1 human cells facilitated formation of extensive tracks of RPA coated 5’ C-rich telomeric single-stranded (ss)DNA, an observation also supported by minimal dependence on conventional end-processing exonucleases MRE11, EXO1, or Apollo. Thus, telomeric DSBs in G1 human cells initiate an abbreviated DDR that results in extensive resection in the absence of 53BP1, facilitating formation of 5’ C-rich overhangs, a previously proposed marker of the recombination dependent, alternative lengthening of telomeres (ALT) pathway, which presumably persist until recombination-mediated elongation and restoration is possible. |
