Human Replication Factor C Stimulates Flap Endonuclease 1

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.

Sulfolobus Replication Factor C Stimulates the Activity of DNA Polymerase B1

“…Some suggested that RFC disengages from DNA after PCNA loading (36)(37)(38), whereas others found that RFC remains attached to the primer terminus and moves along the DNA with PCNA and Pol␦ throughout chain elongation (39,40). The latter scenario appears to be consistent with the finding that RFC interacted with FEN1 and ligase I, two proteins participating in Okazaki fragment maturation (26,27). As it moves along the DNA during the fork progression, RFC would be readily available for stimulating FEN1 in flap cleavage and participating in the sealing of nicks between Okazaki fragments.…”

Esc2 promotes Mus81 complex-activity via its SUMO-like and DNA binding domains

“…The Mus81 complex and other nucleases involved in DNA replication and/or repair, including Rad27 and Rad1-Rad10, are stimulated and targeted by a diverse array of repair and replication proteins. Examples include PCNA and RFC-complex, which stimulate Rad27 (61–63) as well as Srs2 and Rad54 promoting the activity of the Mus81 complex (34,64,65). Mechanistically, these stimulatory factors interact with the particular nuclease, resulting in formation of a complex that cleaves the substrate more efficiently than the nuclease alone, pointing to a requirement for the specific interaction domain on the stimulating protein.…”

The Trans-autostimulatory Activity of Rad27 Suppresses dna2 Defects in Okazaki Fragment Processing

“…Preparation of Substrates-All DNA substrates used in this study were prepared as previously reported (36). Briefly, one of the two oligonucleotides in a partial duplex DNA substrate was 5Ј-labeled with [␥-…”

“…According to the three-dimensional structure of hFEN1-substrate complex (26), the C-terminal region (amino acids from 336 to 380) appears to contain a naturally unstructured extension that may participate in substrate binding, nuclear localization, and protein-protein interactions (22,40,(42)(43)(44). Recently, we showed that human RFC stimulated the endonuclease activity of wild type hFEN1 and this activation depends on the C-terminal region of hFEN1 (36). To confirm the importance of the C terminus of Rad27 and hFEN1 for autostimulation, we constructed expression vectors for three C-terminal truncated versions of Rad27, which included Rad27(1-366), Rad27(1-350), and Rad27(1-338) (16-, 32-, and 44-amino acid deletion, respectively, from the C terminus) and purified each mutant protein as well as the wild type.…”