Alternative Lengthening of Telomeres Mediated by Mitotic DNA Synthesis Engages Break-Induced Replication Processes

ABSTRACT

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. By analyzing telomerase-positive cells and their human TERC knockout-derived ALT human cell lines, we show that ALT cells harbor more fragile telomeres representing telomere replication problems. ALT-associated replication defects trigger mitotic DNA synthesis (MiDAS) at telomeres in a RAD52-dependent, but RAD51-independent, manner. Telomeric MiDAS is a conservative DNA synthesis process, potentially mediated by break-induced replication, similar to type II ALT survivors in Saccharomyces cerevisiae. Replication stresses induced by ectopic oncogenic expression of cyclin E, G-quadruplexes, or R-loop formation facilitate the ALT pathway and lead to telomere clustering, a hallmark of ALT cancers. The TIMELESS/TIPIN complex suppresses telomere clustering and telomeric MiDAS, whereas the SMC5/6 complex promotes them. In summary, ALT cells exhibit more telomere replication defects that result in persistent DNA damage responses at telomeres, leading to the engagement of telomeric MiDAS (spontaneous mitotic telomere synthesis) that is triggered by DNA replication stress, a potential driver of genomic duplications in cancer.

KEYWORDS:

• telomere
• ALT
• telomerase
• MiDAS
• G quadruplex
• R-loop
• cyclin E
• TIMELESS
• SMC5/6
• RAD52
• RAD51

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/MCB.00226-17.

ACKNOWLEDGMENTS

We thank Hongtao Yu (UT Southwestern Medical Center) for sharing reagents and equipment, Eunhee Choi (UT Southwestern Medical Center) for flow cytometric analysis of the cell cycle, and Emily Binshtok for editing and discussion.

This work was supported by grant AG01228 from the National Institute on Aging (W.E.W. and J.W.S.) and by the NCI T32 Cancer Research Training Program (CA124334) (J.M.). We also acknowledge the Harold Simmons NCI Designated Comprehensive Cancer Center Support Grant (CA142543) and the Southland Financial Corporation Distinguished Chair in Geriatric Research (J.W.S. and W.E.W.). This work was performed in laboratories constructed with support from NIH grant C06 RR30414.

We have no conflicts of interest to declare.