FEN1 Ensures Telomere Stability by Facilitating Replication Fork Re-initiation

Julien P. Duxin,Abhishek Saharia,Sheila A. Stewart,Daniel C. Teasley,Benjamin Dao,Katherine B. Chiappinelli

doi:10.1074/jbc.m110.112276

Julien P. Duxin, Abhishek Saharia + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.112276

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Abstract

Telomeres are terminal repetitive DNA sequences whose stability requires the coordinated actions of telomere-binding proteins and the DNA replication and repair machinery. Recently, we demonstrated that the DNA replication and repair protein Flap endonuclease 1 (FEN1) is required for replication of lagging strand telomeres. Here, we demonstrate for the first time that FEN1 is required for efficient re-initiation of stalled replication forks. At the telomere, we find that FEN1 depletion results in replicative stress as evidenced by fragile telomere expression and sister telomere loss. We show that FEN1 participation in Okazaki fragment processing is not required for efficient telomere replication. Instead we find that FEN1 gap endonuclease activity, which processes DNA structures resembling stalled replication forks, and the FEN1 interaction with the RecQ helicases are vital for telomere stability. Finally, we find that FEN1 depletion neither impacts cell cycle progression nor in vitro DNA replication through non-telomeric sequences. Our finding that FEN1 is required for efficient replication fork re-initiation strongly suggests that the fragile telomere expression and sister telomere losses observed upon FEN1 depletion are the direct result of replication fork collapse. Together, these findings suggest that other nucleases compensate for FEN1 loss throughout the genome during DNA replication but fail to do so at the telomere. We propose that FEN1 maintains stable telomeres by facilitating replication through the G-rich lagging strand telomere, thereby ensuring high fidelity telomere replication.

Highlights

Deficiencies in various DNA replication/repair mechanisms become detrimental in highly repetitive DNA sequences that present unique challenges to the DNA replication machinery [4, 5]
Replication fork pausing and stalling occur within telomeric repeats [7,8,9,10], and telomeres were recently identified as fragile sites [11, 12]
In telomerase-positive cells neither telomere dysfunction nor cytogenetic abnormalities were observed upon Flap endonuclease 1 (FEN1) depletion [25]

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—All cells were grown as reported [25,26,27,28]. Briefly, cells were grown at 37 °C in 5% CO2. The FEN1 cDNAs were recombined into the pAdEasy-1 plasmid (Stratagene), and the resultant DNA was transfected into HEK293 cells to produce infectious adenovirus. SV-40 Large-T Antigen-dependent in Vitro DNA Replication Assay—The crude cell extracts for this assay were prepared using HeLa cells as described [31]. Each 25-␮l reaction contained 30 mM HEPES-HCl, pH 7.8, 7 mM MgCl2, 4 mM ATP, 200 ␮M each of CTP, UTP, and GTP, 100 ␮M each of dATP, dGTP, and dTTP, 0.5 mM DTT, 40 mM creatine phosphate, 0.625 units of creatine phosphokinase, 50 ␮M (2.5 ␮Ci) [␣-32P]dCTP (PerkinElmer Life Sciences), 50 ng of linearized plasmid DNA, 1. To verify that the products were generated by semi-conservative replication, additional samples were digested after precipitation with 10 units of DpnI (New England Biolabs) for 5 min at 37 °C, which completely degraded the methylated plasmid template. Statistical Analysis—Student’s t test (two-tailed distribution with equal variance) was used for BrdU foci, CO-FISH, and fragile telomere analyses

RESULTS

Because the mED mutant processes

DISCUSSION