Extrachromosomal Telomeres Derived from Excessive Strand Displacements

Abstract

Alternative Lengthening of Telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication (BIR), evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), unique to ALT cells, are considered potential precursors of C-circles, their generation process remains undefined. Here, we introduce a highly sensitive method to detect single stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single stranded DNAs are near 500 bp in size. Both C-rich ssDNAs and C-circles are abundant in cytoplasmic fraction which supports the idea that C-rich ssDNAs may indeed precede C-circles. We also found that C-rich ssDNAs originate during Okazaki fragment processing in lagging strands. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha)-complex mediated priming of the C-strand synthesis and subsequent excessive strand displacement of C-rich strand through DNA Polymerase delta and BLM helicase. Our work proposes a new model for the generation of C-rich ssDNAs and C-circles that are indicative of the presence of ALT pathway.