Abstract
Human flap endonuclease 1 (FEN1), an essential DNA replication protein, cleaves substrates with unannealed 5'-tails. FEN1 apparently tracks along the flap from the 5'-end to the cleavage site. Proliferating cell nuclear antigen (PCNA) stimulates FEN1 cleavage 5-50-fold. To determine whether tracking, binding, or cleavage is enhanced by PCNA, we tested a variety of flap substrates. Similar levels of PCNA stimulation occur on both a cleavage-sensitive nicked substrate and a less sensitive gapped substrate. PCNA stimulates FEN1 irrespective of the flap length. Stimulation occurs on a pseudo-Y substrate that exhibits upstream primer-independent cleavage. A pseudo-Y substrate with a sequence requiring an upstream primer for cleavage was not activated by PCNA, suggesting that PCNA does not compensate for substrate features that inhibit cleavage. A biotin.streptavidin conjugation at the 5'-end of a flap structure prevents FEN1 loading. The addition of PCNA does not restore FEN1 activity. These results indicate that PCNA does not direct FEN1 to the cleavage site from solution. Kinetic analyses reveal that PCNA can lower the K(m) for FEN1 by 11-12-fold. Overall, our results indicate that after FEN1 tracks to the cleavage site, PCNA enhances FEN1 binding stability, allowing for greater cleavage efficiency.
Highlights
Flap endonuclease 1 (FEN1)1 is a member of the RAD2 superfamily of nucleases that play a critical role in DNA replication and repair in prokaryotes and eukaryotes [1,2,3,4,5]
Proliferating cell nuclear antigen (PCNA) Stimulates FEN1 on a Gapped Substrate—Previous studies have demonstrated that PCNA stimulates FEN1 cleavage activity on flap substrates [13, 20]
PCNA would stimulate FEN1 cleavage activity on a gapped substrate to the level observed for a nicked substrate with PCNA
Summary
Materials—All oligonucleotides were synthesized by Genosys Biotechnologies (The Woodlands, TX). Substrates were annealed by mixing 0.5 pmol of the respective downstream primer with 2 pmol of the corresponding template in annealing buffer (10 mM Tris base, 50 mM KCl, and 1 mM EDTA (pH 8.0)) to a final volume of 50 l. Enzyme Assay—Reactions containing the indicated amounts of substrate, FEN1, and PCNA were performed in a buffer containing 30 mM HEPES (pH 7.6), 5% glycerol, 40 mM KCl, 0.1 mg/ml bovine serum albumin, and 8 mM MgCl2. The initial velocity was determined by measuring the substrate and product intensities on a 12% polyacrylamide, 7 M urea denaturing gel by means of PhosphorImager (Molecular Dynamics) analysis. Gel Mobility Shift Assay—Reactions were performed in a buffer containing 30 mM HEPES (pH 7.6), 5% glycerol, 40 mM KCl, 0.1 mg/ml bovine serum albumin, and 8 mM CaCl2 in a final reaction volume of 20 l. After separation on a 0.7% agarose gel in 0.1ϫ TBE (8.9 mM Tris base, 8.9 mM boric acid, and 0.2 mM EDTA (pH 8.0)), products were visualized using a PhosphorImager (Molecular Dynamics)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
