Investigating the role of DNA polymerase theta in telomeric stability and the escape from crisis

During a telomere crisis, critically shortened telomeres are recognised as DNA double-strand breaks (DSBs) by the DNA damage response apparatus, resulting in telomere fusions which propagate genomic instability. DNA polymerase theta (POLQ) is a crucial component of the alternative non-homologous end-joining (A-NHEJ) repair pathway that is implicated in the formation of intra-chromosomal telomere fusion events. Crisis escape is achieved when telomere lengths stabilise following the reestablishment of telomerase activity, as a consequence of large-scale genomic rearrangements. Thus, POLQ is potentially important in malignant transformation and is over-expressed in diverse cancers and associated with poor clinical outcome. This study aimed to investigate the impact of POLQ on the capacity of cells to escape a telomere-driven crisis, through exploring the role of POLQ in NHEJ repair mechanisms utilised during crisis and its impact on genome evolution. The disruption of POLQ in two distinct human cell lines resulted in a shorter crisis interval compared with their wild-type (WT) counterparts. A significant increase in inter-chromosomal telomere fusion events was observed for the POLQ-deficient cell lines compared with the WT cell lines. This difference was confirmed for one cell line using high-resolution Illumina paired-end sequencing of crisis-stage fusion amplicons. Conversely, a notable reduction in intra-chromosomal 17p sister chromatid fusions was determined for the POLQ-deficient compared with the WT HCT116 cell line. The differences in inter/intra-chromosomal telomere fusion events was reproduced in experiments employing targeted nucleases to induce telomere specific DSBs. Whole genome sequencing of WT and POLQ-deficient clones before and after crisis revealed the remarkable heterogeneity of POLQ-deficient clones that successfully traversed crisis. Taken together, these data reveal the essential role that POLQ-dependent A-NHEJ plays in mediating intra chromosomal telomere fusion and the escape from crisis. These observations may have implications for the use of POLQ as a therapeutic target