Abstract
Flap endonuclease 1 (FEN1) is the enzyme responsible for specifically removing the flap structure produced during DNA replication, repair, and recombination. Here we report that the human replication factor C (RFC) complex stimulates the nuclease activity of human FEN1 in an ATP-independent manner. Although proliferating cell nuclear antigen is also known to stimulate FEN1, less RFC was required for comparable FEN1 stimulation. Kinetic analyses indicate that the mechanism by which RFC stimulates FEN1 is distinct from that by proliferating cell nuclear antigen. Heat-denatured RFC or its subunit retained, fully or partially, the ability to stimulate FEN1. Via systematic deletion analyses, we have defined three specific regions of RFC4 capable of stimulating FEN1. The region of RFC4 with the highest activity spans amino acids 170-194 and contains RFC box VII. Four amino acid residues (i.e. Tyr-182, Glu-188, Pro-189, and Ser-192) are especially important for FEN1 stimulatory activity. Thus, RFC, via several stimulatory motifs per molecule, potently activates FEN1. This function makes RFC a critical partner with FEN1 for the processing of eukaryotic Okazaki fragments.
Highlights
Faithful genome maintenance is fundamental to the preservation of life
We report for the first time that the human replication factor C (RFC) complex markedly stimulates human Flap endonuclease 1 (FEN1) via its multiple stimulatory motifs per subunit, empowering FEN1 to cleave more efficiently all its known substrates
FEN1 stimulation by RFC is independent of ATP binding and hydrolysis, suggesting that this function of RFC is novel and separable from ATP-dependent proliferating cell nuclear antigen (PCNA) loading to primer ends
Summary
Enzymes and Nucleotides—The oligonucleotides used in this study were commercially synthesized from Genotech (Daejeon, Korea), and their sequences are listed in Table 1. [␥-32P]ATP (3,000 Ci/mmol) was purchased from Amersham Biosciences and Izotop (Budapest, Hungary). For this purpose, we tested effects of increasing the presence of RFC, the amounts of products formed concentrations (2, 5, 10, 15, and 20 mM) of Mg2ϩ on FEN1 were 9.3 fmol (ϳ30-fold more than in its absence) after 30 min activity in the presence of increasing levels (0, 5, and 20 fmol) of of incubation (Fig. 2, A and B). 2.5, 5, 20 fmol) of RFC increased the rate of FEN1-catalyzed lower levels (5 fmol) of RFC stimulated FEN1 activity, most cleavage (Fig. 2C) This kinetic analysis revealed that the effectively in Mg2ϩ concentrations less than 10 mM (Fig. 1D). This result indicates that stimulation of FEN1 by RFC occurs independently of the RFC loading function of PCNA
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