Extrachromosomal telomere DNA derived from excessive strand displacements

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 121; no. 19; p. e2318438121
• Main Authors: Lee, Junyeop, Lee, Jina, Sohn, Eric J., Taglialatela, Angelo, O’Sullivan, Roderick J., Ciccia, Alberto, Min, Jaewon
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 07.05.2024
• Subjects: Biological Sciences; Blotting, Southern; Cancer; Cytoplasm; Deoxyribonucleic acid; DNA; DNA - genetics; DNA - metabolism; DNA biosynthesis; DNA helicase; DNA polymerase; DNA Polymerase III - genetics; DNA Polymerase III - metabolism; DNA Replication; DNA, Circular - genetics; DNA, Circular - metabolism; DNA, Single-Stranded - genetics; DNA, Single-Stranded - metabolism; DNA-directed DNA polymerase; Humans; Nucleotides; Primase; Single-stranded DNA; Synthesis; Telomere - genetics; Telomere - metabolism; Telomere Homeostasis; Telomeres; POLD3; C-circle; BLM; CST; break-induced replication
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.2318438121

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication, 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), including C-circles, are unique to ALT cells, their generation process remains undefined. Here, we introduce a 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 that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear and circular C-rich ssDNAs are generated concurrently. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation.