Molecular steps of G-overhang generation at human telomeres and its function in chromosome end protection

Abstract

Telomeric G-overhangs are required for the formation of the protective telomere structure and telomerase action. However, the mechanism controlling G-overhang generation at human telomeres is poorly understood. Here, we show that G-overhangs can undergo cell cycle-regulated changes independent of telomerase activity. G-overhangs at lagging telomeres are lengthened in S phase and then shortened in late S/G2 because of C-strand fill-in, whereas the sizes of G-overhangs at leading telomeres remain stable throughout S phase and are lengthened in G2/M. The final nucleotides at measurable C-strands are precisely defined throughout the cell cycle, indicating that C-strand resection is strictly regulated. We demonstrate that C-strand fill-in is mediated by DNA polymerase a (pola) and controlled by cyclin-dependent kinase 1 (CDK1). Inhibition of CDK1 leads to accumulation of lengthened G-overhangs and induces telomeric DNA damage response. Furthermore, depletion of hStn1 results in elongation of G-overhangs and an increase in telomeric DNA damage. Our results suggest that G-overhang generation at human telomeres is regulated by multiple tightly controlled processes and C-strand fill-in is under the control of pola and CDK1.

For cancers there is more to life than a longer G-strand

“…Three recent studies from the Wright [1], Chai [2] and de Lange [3] labs have elucidated previously neglected aspects of cancer cell te lo mere maintenance. Because inhibiting telomere maintenance can selectively kill cancer cells without harming normal cells [4,5], this new understanding of the mechanism a n d d y n a m i c s i n v o l v e d m a y provide useful new strategies for treating cancer.…”

“…These studies were performed in cell cycle synchronized and telo merase + human cell lines. Telomerase did not appear essential for the general overhang length dynamics [2]. Wu et al [3] further contributed by using knock-out mouse models to study the role of Apollo nuclease.…”

“…Leading-strand replication syn thesized a new G-strand to the end of the C-strand template [2], creating a blunt end. The daughter G-strand was shorter than the parental G-strand because the template was shorter (by 60 nt).…”

“…Apollo immediately resected the C-strand to provide t h e o v e r h a n g t h a t t e l o m e r a s e requires. Telomerase then elongated the G-strand on some telomeres until it overhung the C-strand by > 70 nt [1][2][3]. However, many leading telomeres may not have been appreciably elongated [2].…”

“…Telomerase then elongated the G-strand on some telomeres until it overhung the C-strand by > 70 nt [1][2][3]. However, many leading telomeres may not have been appreciably elongated [2]. In late S-phase, the leading telomeres with longer overhangs had their C-strands elongated to reduce the overhang length back to 60 nt [1,2].…”