Efficient and faithful replication of telomeric DNA is critical for maintaining genome integrity. The G-quadruplex (G4) structure arising in the repetitive TTAGGG sequence is thought to stall replication forks, impairing efficient telomere replication and leading to telomere instabilities. However, pathways modulating telomeric G4 are poorly understood, and it is unclear whether defects in these pathways contribute to genome instabilities in vivo. Here, we report that mammalian DNA2 helicase/nuclease recognizes and cleaves telomeric G4 in vitro. Consistent with DNA2's role in removing G4, DNA2 deficiency in mouse cells leads to telomere replication defects, elevating the levels of fragile telomeres (FTs) and sister telomere associations (STAs). Such telomere defects are enhanced by stabilizers of G4. Moreover, DNA2 deficiency induces telomere DNA damage and chromosome segregation errors, resulting in tetraploidy and aneuploidy. Consequently, DNA2-deficient mice develop aneuploidyassociated cancers containing dysfunctional telomeres. Collectively, our genetic, cytological, and biochemical results suggest that mammalian DNA2 reduces replication stress at telomeres, thereby preserving genome stability and suppressing cancer development, and that this may involve, at least in part, nucleolytic processing of telomeric G4.

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.

Histone deacetylase 3 (HDAC3) contributes to the regulation of gene expression, chromatin structure, and genomic stability. Because HDAC3 associates with oncoproteins that drive leukemia and lymphoma, we engineered a conditional deletion allele in mice to explore the physiological roles of Hdac3 in hematopoiesis. We used the Vav-Cre transgenic allele to trigger recombination, which yielded a dramatic loss of lymphoid cells, hypocellular bone marrow, and mild anemia. Phenotypic and functional analysis suggested that Hdac3 was required for the formation of the earliest lymphoid progenitor cells in the marrow, but that the marrow contained 3-5 times more multipotent progenitor cells. Hdac3 -/-stem cells were severely compromised in competitive bone marrow transplantation. In vitro, Hdac3 -/-stem and progenitor cells failed to proliferate, and most cells remained undifferentiated. Moreover, one-third of the Hdac3 -/-stem and progenitor cells were in S phase 2 hours after BrdU labeling in vivo, suggesting that these cells were impaired in transit through the S phase. DNA fiber-labeling experiments indicated that Hdac3 was required for efficient DNA replication in hematopoietic stem and progenitor cells. Thus, Hdac3 is required for the passage of hematopoietic stem/progenitor cells through the S phase, for stem cell functions, and for lymphopoiesis.

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...