Human Flap Endonuclease I Is in Complex with Telomerase and Is Required for Telomerase-mediated Telomere Maintenance

Abstract

Studies from budding yeast and ciliates have suggested that telomerase extension of telomeres requires the conventional DNA replication machinery, yet little is known about how DNA replication proteins regulate telomerase action in higher eukaryotic cells. Here we investigate the role of one of the DNA replication factors, flap endonuclease I (FEN1), in regulating telomerase activity in mammalian cells. FEN1 is a nuclease that plays an important role in DNA replication, repair, and recombination. We show that FEN1 is in complex with telomerase in vivo via telomeric DNA. We further demonstrate that FEN1 deficiency in mouse embryonic fibroblasts leads to an increase in telomere end-to-end fusions. In cancer cells, FEN1 deficiency induces gradual shortening of telomeres but does not alter the single-stranded G-overhangs. This is, to our knowledge, the first evidence that FEN1 and telomerase physically coexist as a complex and that FEN1 can regulate telomerase activity at telomeres in mammalian cells.Telomeres are distinct DNA-protein structures that protect eukaryotic chromosome ends from degradation and inappropriate recombination or fusions. Maintenance of functional telomeres is essential for long term cell proliferation and stem cell self-renewal. In normal human somatic cells, telomeres progressively shorten each time a cell divides (1). When a subset of telomeres becomes critically short, these short telomeres are recognized as damaged DNA (1-6). Activation of the DNA damage response pathway then induces cellular senescence, impairing cell proliferation (7-9).To counteract telomere shortening, the cells activate a special reverse transcriptase, telomerase, to elongate short telomeres. Telomerase adds telomeric DNA repeats to chromosome ends, thus keeping telomeres functional (10, 11). Indeed, telomerase is up-regulated in cells that need long term proliferation potential such as embryonic stem cells, germline cells, cancer stem cells, activated lymphocytes, and the majority of human cancer cells (12-16). The critical roles of telomerase in tumor proliferation and stem cell behavior underscore the importance of understanding the regulatory mechanisms for telomerase action at telomeres.Telomerase elongation of telomeres is a highly coordinated and tightly regulated process, so that the length of the telomeric repeats is kept within a cell type-specific narrow range from 3 to 20 kb in human cells (17). Telomere homeostasis is maintained by a number of proteins associated with the telomere and/or telomerase. These proteins control the recruitment and accessibility of telomerase to telomeres and regulate telomerase activities at telomeres. Defects in certain proteins, among which are DNA metabolic proteins, have been shown to positively or negatively influence telomere length (18).Several studies in yeast and ciliates have suggested that telomerase-dependent telomere extension is coupled with conventional DNA replication and requires certain DNA replication proteins. For example, inactivation of components of budd...

Prospectively isolated CD133/CD24‐positive ependymal cells from the adult spinal cord and lateral ventricle wall differ in their long‐term in vitro self‐renewal and in vivo gene expression

“…Interestingly, we also found two genes, Fen1 and Rtel1, which are critical for telomere stability and maintenance of dividing cells (Ding et al, 2004;Sampathi et al, 2009). The presence of three of these genes, Efnb1 (encoding Ephrin-B1), Fen1, and Ret was confirmed on the protein level in SC ependymal cells (Fig.…”

“…We found the genes Rtel and Fen1, coding for proteins that are essential for chromosomal stability and telomere maintenance, to be higher expressed by SC ependymal cells, neurospheres derived from them, and by radial glial cells, indicating that these cells are molecularly equipped to long-term self-renew (Ding et al, 2004;Sampathi et al, 2009). In addition, we identified a large number of RA-responsive genes higher expressed by SC ependymal cells and surprisingly found RA to act on SC ependymal cells by increasing their cell number in vitro.…”

The human Suv3 helicase interacts with replication protein A and flap endonuclease 1 in the nucleus

“…hSuv3 could potentially modulate the activity of FEN1 in vivo in one of its many functions in DNA repair, telomere maintenance, Okazaki fragment maturation or resolving stalled replication forks [46,47]. In this case, hSuv3 was shown to stimulate the incision activity of FEN1, independently of flap length and only in part on helicase activity.…”

CHIP Promotes Human Telomerase Reverse Transcriptase Degradation and Negatively Regulates Telomerase Activity

“…We repeatedly treated H1299 cells with CHIP siRNA at 3-day intervals for 2 weeks. The same method has been used to study the role of the DNA replication factors in telomere maintenance (38,39). Whereas about 80 -90% of endogenous CHIP was depleted during the course of repetitive siRNA transfection (supplemental Fig.…”