A novel role in DNA metabolism for the binding of Fen1/Rad27 to PCNA and implications for genetic risk.

Polymorphisms occur throughout the human genome. In some cases, they may affect biological function and may be responsible for poorly understood differences in individual susceptibilities to diseases. Because genetic instability is a major causative factor in tumorigenesis, the consequences of subtle functional alterations in proteins that act on DNA are of particular interest. The Fen1 nuclease, termed Rad27 in the yeast Saccharomyces cerevisiae, plays an important role in DNA metabolism (reviewed in reference 24). It seems plausible that even subtle changes in Fen1 could have significant deleterious effects because deletion of the Fen1/Rad27 gene in yeast cells causes severe effects, including temperature sensitivity and hypersensitivity to the DNA-alkylating agent methyl methanesulfonate (MMS). The rad27-Δ (a deletion null allele) strain exhibits instabilities in short DNA repeats, microsatellites and minisatellites, expansions of the trinucleotide repeat CTG/CAG, duplications between separated short direct random repeats, intrachromosomal and interchromosomal mitotic recombination, and chromosome loss (8, 15, 20, 38, 41, 44, 48). RAD27 becomes essential for viability in strains lacking double-strand break (DSB) recombinational repair (42, 44) or the 5′→3′ exonuclease EXO1 (43) or in strains carrying mutation pol3-01 in the 3′→5′ exonuclease proofreading domain of DNA polymerase δ (Pol δ) (20), and in strains with a temperature-sensitive mutation in DNA2 (DNA helicase/3′→5′ exonuclease) (1, 3). The Fen1 nuclease recognizes specific types of DNA structures (reviewed in reference 24). In vitro, it is highly active toward 5′-flap DNA, a branched structure that can result from strand displacement during DNA synthesis. Fen1 acts as an endonuclease to cleave the displaced flap strand at the single-strand/double-strand junction. It also acts as a 5′→3′ exonuclease at nicks in duplex DNA. Significantly, Fen1 can remove a 5′-terminal ribonucleotide as well. During lagging strand DNA synthesis, RNA primers are removed by RNase H1; however, this enzyme cannot excise the final 5′-terminal ribonucleotide at the RNA-DNA junction. The completion of RNA primer removal by Fen1 is essential for Okazaki fragment processing in reconstituted replication assays (14, 35, 47, 49). At the restrictive temperature (37°C), a rad27-Δ mutant accumulates short DNA fragments of the size range expected for unprocessed Okazaki fragments (27). Fen1 can cleave oligonucleotide substrates with 5′-terminal abasic sites that mimic reaction intermediates that arise during base excision repair (BER) (6, 32). In reconstituted long-patch BER assays, Fen1 is essential for the excision step (18, 19). The relevance of these in vitro observations to BER in vivo is supported by the MMS sensitivity of rad27-Δ mutants. In addition to its structure-specific nuclease activity, Fen1 exhibits a protein-protein interaction with proliferating cell nuclear antigen (PCNA). PCNA is essential for DNA synthesis during replication and repair (reviewed in references 17 and 50). It is a homotrimeric ring-shaped protein that serves as an accessory factor for Pol δ and Pol ɛ. DNA-bound PCNA forms a sliding clamp that tethers Pol δ or Pol ɛ to template DNA and thus promotes processive DNA synthesis. It also binds to several other proteins involved in DNA metabolism, including DNA ligase I (22) and DNA-(cytosine-5) methyltransferase (4). PCNA binds to Fen1 and stimulates nuclease activity on oligonucleotide-based 5′ flaps and nicked duplex DNA substrates (23, 54). These properties suggest that Fen1 and PCNA interact during the course of DNA replication, DNA repair, or both. Support for the importance of Fen1-PCNA binding in BER has come from studies with a reconstituted long-patch BER system, in which Fen1-PCNA interaction was shown to contribute to excision efficiency (9). The possible involvement of the Fen1-PCNA interaction in DNA replication has been proposed (23), but it has not been investigated specifically. Recently, a hypothetical model of the Fen1-PCNA complex was presented that illustrated PCNA binding and nuclease activities working in tandem during replication (13). We have selectively inactivated nuclease and PCNA binding activities of yeast Fen1/Rad27 to dissect the contributions of each to overall function in vivo. Unexpectedly, we found that PCNA binding in Rad27/Fen1 has a function beyond mere stimulation of nuclease activity. A mutation of Rad27 eliminating PCNA binding had very little effect by itself but had major consequences via intragenic or intergenic interactions. The PCNA nonbinding rad27 allele greatly enhanced the repair deficiency of a rad51-null strain and caused inviability of yeast cells with a minor Pol δ defect. Our results show that while subtle defects in specific DNA metabolic proteins may have little impact on their own, they can result in severe phenotypic effects in combination. This principle, when applied to naturally occurring polymorphisms, can have profound implications for the inheritance of susceptibility to disease.