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Human replication factor C stimulates flap endonuclease 1.
Il-Taeg Cho1, Do-Hyung Kim, Young-Hoon Kang
1Center for DNA Replication and Genome Instability, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.
The Journal of Biological Chemistry
|February 12, 2009
Summary
The replication factor C (RFC) complex enhances the flap endonuclease 1 (FEN1) enzyme activity, crucial for DNA replication and repair. This RFC-FEN1 interaction is ATP-independent and vital for processing Okazaki fragments in eukaryotes.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Flap endonuclease 1 (FEN1) is essential for removing flap structures during DNA replication, repair, and recombination.
- FEN1 activity is known to be stimulated by proliferating cell nuclear antigen (PCNA).
Purpose of the Study:
- To investigate the effect of the human replication factor C (RFC) complex on human FEN1 nuclease activity.
- To elucidate the mechanism and identify key regions of RFC involved in FEN1 stimulation.
Main Methods:
- In vitro assays measuring FEN1 nuclease activity.
- Kinetic analyses comparing RFC and PCNA stimulation.
- Systematic deletion analyses of RFC subunit RFC4.
- Site-directed mutagenesis to identify critical amino acid residues.
Main Results:
- Human RFC complex stimulates human FEN1 activity in an ATP-independent manner.
- RFC requires lower concentrations than PCNA for comparable FEN1 stimulation.
- Specific regions within RFC4, particularly amino acids 170-194 (RFC box VII) and residues Tyr-182, Glu-188, Pro-189, and Ser-192, are critical for stimulation.
- Heat-denatured RFC or its subunits retain partial FEN1 stimulatory ability.
Conclusions:
- RFC potently activates FEN1 through multiple stimulatory motifs.
- RFC is a critical partner for FEN1 in processing eukaryotic Okazaki fragments.
- The mechanism of RFC-mediated FEN1 stimulation is distinct from that of PCNA.
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