Functional Analysis of Human FEN1 in Saccharomyces Cerevisiae and Its Role in Genome Stability

Abstract

The flap endonuclease, FEN1, is an evolutionarily conserved component of DNA replication from archaebacteria to humans. Based on in vitro results, it processes Okazaki fragments during replication and is involved in base excision repair. FEN1 removes the last primer ribonucleotide on the lagging strand and it cleaves a 5' flap that may result from strand displacement during replication or during base excision repair. Its biological importance has been revealed largely through studies in the yeast Saccharomyces cerevisiae where deletion of the homologous gene RAD27 results in genome instability and mutagen sensitivity. While the in vivo function of Rad27 has been well characterized through genetic and biochemical approaches, little is understood about the in vivo functions of human FEN1. Guided by our recent results with yeast RAD27, we explored the function of human FEN1 in yeast. We found that the human FEN1 protein complements a yeast rad27 null mutant for a variety of defects including mutagen sensitivity, genetic instability and the synthetic lethal interactions of a rad27 rad51 and a rad27 pol3-01 mutant. Furthermore, a mutant form of FEN1 lacking nuclease function exhibits dominant-negative effects on cell growth and genome instability similar to those seen with the homologous yeast rad27 mutation. This genetic impact is stronger when the human and yeast PCNA-binding domains are exchanged. These data indicate that the human FEN1 and yeast Rad27 proteins act on the same substrate in vivo. Our study defines a sensitive yeast system for the identification and characterization of mutations in FEN1.

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

“…For instance, Resnick and colleagues (25,26) have used a FEN1 mutant that harbors a D to A substitution at a residue found to be critical for DNA processing to determine the in vivo substrates of the flap endonuclease. Using either genetically engineered yeast strains or exogenously expressing yeast systems, they show that the FEN1 mutant causes genetic instability and increased sensitivity to MMS, likely by preventing processing of DNA replication intermediates and promoting the formation of recombinogenic DNA double-strand breaks.…”

“…For example, such a mutation in the flap endonuclease, FEN1 (i.e., a D to A substitution), resulted in a dominant-negative form of the human and yeast protein that bound substrate DNAwith high affinity and blocked subsequent nucleic acid processing in vivo (25,26). Prior studies have established the importance of the acidic residues E96 and D210 to the enzymatic repair activity of APE1 ( Fig.…”

Human ChlR1 Stimulates Endonuclease Activity of hFen1 Independently of ATPase Activity

“…Failure of Okazaki fragment processing in yeast is associated with increasing DNA double-strand breaks, higher mutation rates and generation of genome instabilities. 7,8 Efficient processing of the Okazaki fragment is therefore essential for DNA replication and cell cycle progression. Human Fen1 protein (hFen1) can cleave the flap structure at the junction between ssDNA and dsDNA, an intermediate formed during Okazaki fragment processing, resulting in ligatable nicked dsDNA.…”

Saccharomyces cerevisiae RAD27 complements its Escherichia coli homolog in damage repair but not mutation avoidance

“…Recent studies have shown that the expression of human Fen-1 protein in yeast rad27 D cells can correct a number of mutant phenotypes, including sensitivity to MMS and a high level of spontaneous mutation (Hansen et al 2000;Greene et al 1999), and that expression of the 5' 3' exonuclease domain of E. coli PolI in yeast rad27 D cells could complement sensitivity to MMS and mutator phenotypes (Sun et al 2002). In these cases, however, the extent of the complementation was partial.…”

“…In these cases, however, the extent of the complementation was partial. The inability of human Fen-1 protein or E. coli PolI protein to completely complement the sensitivity to MMS or mutation avoidance in yeast rad27 D may be a result of differences in the ability of the Fen-1 protein or PolI protein and the Rad27 protein to undergo interaction and stimulation by the yeast PCNA protein, a platform for replication and repair (Hansen et al 2000;Greene et al 1999). Thus, human Fen-1, yeast Rad27 and E. coli PolI 5' 3' exonuclease act on the same substrate, but there are differences in interaction between PCNA (or bacterial b -clamp) and Fen-1/Rad27/PolI.…”