C-Terminal Flap Endonuclease (rad27) Mutations: Lethal Interactions With a DNA Ligase I Mutation (cdc9-p) and Suppression by Proliferating Cell Nuclear Antigen (POL30) in Saccharomyces cerevisiae

During lagging-strand DNA replication in eukaryotic cells primers are removed from Okazaki fragments by the flap endonuclease and DNA ligase I joins nascent fragments. Both enzymes are brought to the replication fork by the sliding clamp proliferating cell nuclear antigen (PCNA). To understand the relationship among these three components, we have carried out a synthetic lethal screen with cdc9-p, a DNA ligase mutation with two substitutions (F43A/F44A) in its PCNA interaction domain. We recovered the flap endonuclease mutation rad27-K325* with a stop codon at residue 325. We created two additional rad27 alleles, rad27-A358* with a stop codon at residue 358 and rad27-pX8 with substitutions of all eight residues of the PCNA interaction domain. rad27-pX8 is temperature lethal and rad27-A358* grows slowly in combination with cdc9-p. Tests of mutation avoidance, DNA repair, and compatibility with DNA repair mutations showed that rad27-K325* confers severe phenotypes similar to rad27Δ, rad27-A358* confers mild phenotypes, and rad27-pX8 confers phenotypes intermediate between the other two alleles. High-copy expression of POL30 (PCNA) suppresses the canavanine mutation rate of all the rad27 alleles, including rad27Δ. These studies show the importance of the C terminus of the flap endonuclease in DNA replication and repair and, by virtue of the initial screen, show that this portion of the enzyme helps coordinate the entry of DNA ligase during Okazaki fragment maturation.CELLULAR maintenance of genomic integrity is essential for the continued viability of all organisms. The fidelity of DNA replication has to be maintained and DNA insults have to be repaired to ensure that deleterious mutations are not passed on to progeny or cause cancerous growth. A number of cellular proteins have multiple roles in DNA replication, mutation avoidance, and repair. In Saccharomyces cerevisiae, the flap endonuclease, proliferating cell nuclear antigen (PCNA), and DNA ligase I encoded by RAD27, POL30, and CDC9, respectively, are all required for proper replication and also function to avoid mutation and to facilitate repair.The flap endonuclease, FEN-1 in humans, is a highly conserved structure-specific nuclease that has both endonuclease and 5′–3′ exonuclease activity. During lagging-strand replication these activities function to remove primers from Okazaki fragments, either by endonucleolytic cleavage of a flap made by strand displacement (Liu et al. 2004) or by sequential exonucleolytic removal of single nucleotides at the 5′ end of the primer (Murante et al. 1994).While deletion of RAD27 is not lethal to yeast cells, the rad27Δ mutant exhibits temperature-sensitive growth, is a mutator, and undergoes genomic instability (Johnson et al. 1995; Reagan et al. 1995; Tishkoff et al. 1997b; Chen and Kolodner 1999). In addition, its sensitivity to low doses of the methylating agent methylmethane sulfonate (MMS) implicates the participation of the enzyme in base excision repair (BER) (Reagan et al. 1995; Wu and Wang 1999). rad27Δ mutants have been reported to be either mildly sensitive to UV light or not sensitive to UV light (Reagan et al. 1995; Sommers et al. 1995). In the strain background that the mutant is mildly sensitive, its combination with rad2Δ yields a double mutant more sensitive than each single mutant, implying that the enzyme does not participate in RAD2-mediated nucleotide excision repair (NER) (Reagan et al. 1995). The flap endonuclease has also been implicated in double-strand break (DSB) repair by virtue of the incompatibility of rad27Δ with mutations of the DSB repair pathways (Tishkoff et al. 1997b; Symington 1998). In addition, either the yeast enzyme or its human ortholog has been shown to participate in reactions of homologous recombination, nonhomologous end joining, and telomere maintenance (Parenteau and Wellinger 1999, 2002; Wu et al. 1999; Wang et al. 2004; Kikuchi et al. 2005). Curiously, the rad27Δ mutant is not sensitive to gamma radiation but is sensitive to high doses of MMS that are thought to act as a radiomimetic agent (Reagan et al. 1995; Sommers et al. 1995).PCNA is the replicative clamp that acts as a scaffold to facilitate the loading of DNA replication and repair proteins, including DNA ligase I and the flap endonuclease to DNA (Warbrick 2000, 2006; Maga and Hubscher 2003). PCNA (POL30) is essential for cell viability, which is indicative of its central role in DNA metabolism. Biochemical characterization of its effect on the flap endonuclease shows that it stimulates its activity ∼50-fold, evidencing the productive nature of the interaction (Gomes and Burgers 2000; Tom et al. 2000; Frank et al. 2001; Stucki et al. 2001). The ability of DNA ligase to efficiently catalyze the formation of phosphodiester bonds in the DNA backbone may also be facilitated by its binding to PCNA. Tom et al. (2001) showed that, in vitro, PCNA enhances the ligation reaction 5-fold and that the stable association of DNA ligase with nicked duplex DNA requires PCNA.Both DNA ligase and the flap endonuclease bind to PCNA via their respective PCNA interactive peptide domains (PIP box). The PIP box is a conserved sequence motif of the amino acids QXXLXXFF. The PIP box fits into the interdomain connector loop (IDCL) of PCNA to provide a protein–protein interaction surface (Gomes and Burgers 2000; Chapados et al. 2004; Sakurai et al. 2005; Pascal et al. 2006). Mutations in the PIP box or the IDCL that impair the interaction of DNA ligase and the flap endonuclease to PCNA lead to genomic instability (Amin and Holm 1996; Eissenberg et al. 1997; Gary et al. 1999; Refsland and Livingston 2005; Subramanian et al. 2005). We have reported that the double mutants made by combinations of cdc9-p, rad27-p, and pol30-90—mutations with alterations of the PIP box or the IDCL in the respective proteins—have synergistic phenotypes with respect to MMS sensitivity and to trinucleotide repeat instability (Refsland and Livingston 2005). These results suggest that the two enzymes function in a concerted manner that is facilitated by PCNA.The precise nature of how PCNA coordinates the entry of the flap endonuclease and DNA ligase into the replication fork is not well understood. Biochemical and structural studies have begun to elucidate a possible ordering of these PCNA-mediated interactions. The possibility of such an ordering is underscored by the observation that DNA ligase adopts a toroidal conformation by completely encircling duplex DNA while interacting with PCNA (Pascal et al. 2004). Moreover, both PCNA and DNA ligase may be loaded onto the DNA in a mechanism utilizing the replication clamp loader replication factor C (RFC) (Levin et al. 2004; Vijayakumar et al. 2009), again suggesting a complete encirclement of the DNA by DNA ligase as well as by PCNA. PCNA and DNA ligase are similar in size and their interaction is likely to extend along the face of PCNA in a manner that would prevent other proteins such as the flap endonuclease from binding to the IDCL (Pascal et al. 2004, 2006). A biochemical study with purified yeast proteins showed that the two enzymes cannot bind simultaneously to PCNA (Subramanian et al. 2005). These studies suggest that a coordinated sequential interaction among PCNA, DNA ligase, and the flap endonuclease is important for replication and repair.Alternatively, both the flap endonuclease and DNA ligase may bind to the same molecule of PCNA. Since PCNA is a homotrimer, DNA ligase can potentially bind to one monomer while the flap endonuclease binds to another, using its extended C-terminal tail in a conformation allowing it to be tethered to PCNA concurrently with DNA ligase (Gomes and Burgers 2000; Sakurai et al. 2005). DNA ligase could also bind to PCNA in an extended conformation while the flap endonuclease cleaves the DNA. Sulfolobus solfataricus DNA ligase has been shown to have an open, extended conformation while binding to PCNA (Pascal et al. 2006). Presumably, once the flap endonuclease has removed the 5′ flap, DNA ligase acquires a closed, ring-shaped conformation to catalyze the joining of Okazaki fragments (Pascal et al. 2006).Exactly how the interaction of these enzymes with PCNA is coordinated in vivo, whether singly or concurrently, is not well understood. To further elucidate how the interaction of DNA ligase with PCNA is ordered, we performed a genetic screen to identify mutations that are synthetically lethal with cdc9-p (F44A/F35A), an allele of DNA ligase that has impaired binding to PCNA (Refsland and Livingston 2005; Subramanian et al. 2005). We postulated that genes recovered from this screen would function in DNA repair, replication, and recombination or would be involved in ordering the DNA ligase–PCNA interaction. From the screen we recovered a truncated allele of RAD27, rad27-K325*. This allele encodes a protein that lacks the PIP box and the entire C-terminal domain of the enzyme but retains the N terminus containing the nuclease activities. We have characterized this allele and compared it to two other rad27 alleles in which we have created different alterations of the C-terminal end of the flap endonuclease.