In order to preserve the fidelity of genome duplication during DNA replication, cells with complex genomes have evolved a network of pathways composed of the DNA replication apparatus, DNA repair proteins, and regulatory activities. Despite years of general characterization, knowledge of the specific mechanisms by which these pathways are integrated to protect the genome is still incomplete because of the complexity of underlying replication fork processes and their regulation. The challenge in understanding high fidelity genome transmission has progressed from identification and characterization of the individual DNA replication components to investigation of how they combine to form pathways orchestrating repair and regulation.
The first line of defense against genome instability resides with the enzymes of the DNA replication apparatus itself. While the most familiar example is the proofreading activity found in the DNA polymerases, other proteins of the replisome have also evolved substrate specificities to address errors made during replication fork progression. One of these proteins is Dna2p, a helicase/nuclease. The dna2–1
mutation was identified in a screen for yeast mutants defective in DNA replication based on an assay using permeabilized cells [1
strains were then shown to accumulate subgenomic-size DNA fragments when incubated at the restrictive temperature [2
]. Dna2p has both DNA helicase and single-stranded nuclease activities [3
]. Biochemical and genetic characterization has revealed that Dna2p is involved in the processing of some, but not all, Okazaki fragments. Specifically, it has been proposed that Dna2p acts with FEN1 to remove RNA primers from Okazaki fragments whose 5′ RNA/DNA termini have been extensively displaced by DNA polymerase (pol) δ. Four lines of evidence support a role for Dna2p in Okazaki fragment processing (OFP). First, Dna2p co-purifies with FEN1, which is a structure-specific nuclease required for OFP in the SV40 in vitro replication system [5
]. Second, overexpression of the Saccharomyces cerevisiae
FEN1 gene, RAD27,
suppresses the temperature-sensitive (ts) growth defect of a dna2–1
strain, and furthermore, the dna2–1 rad27
Δ double mutant is synthetically lethal [7
]. Third, biochemical reconstitution experiments have shown that excessive strand displacement by pol δ creates long 5′ flaps that are cleaved inefficiently by FEN1, and that initial cleavage of these flaps by Dna2p potentiates more efficient subsequent cleavage by FEN1 [9
]. Finally, Dna2p prefers to act on flaps with secondary structures in vitro, i.e., hairpins or fold-backs containing CTG repeats, which is probably where helicase functionality becomes necessary [12
]. Thus, the Dna2p nuclease has evolved the ideal mechanism for highly specific action at a replication fork, requiring the presence of an unpaired 5′ terminus for activity, and displaying a complete lack of activity for single-stranded gaps in duplex DNA, which would result in recombinogenic double-strand breaks (DSBs). Nevertheless, there is a real question of whether the excessively displaced flaps used to study Dna2p in vitro ever occur in vivo, and some biochemical evidence suggests this may not be the case. Therefore, although biochemical data indicate that Dna2p is required for OFP when FEN1 or pol δ activity is impaired, identification of Dna2p's in vivo substrates requires more appropriate genetic and physiological assays than have been applied to date [14
The second and third lines of defense for preventing genome instability during DNA replication are the DNA repair pathways and regulatory pathways, such as cell cycle checkpoints. Increasing evidence suggests that these pathways are integrated into the replication pathway through their use of certain replication proteins [15
]. Dna2p seems to be one of these multitasking proteins. Besides its function in OFP, our evidence strongly suggests that Dna2p provides a link between DNA replication and DNA repair, since dna2
mutants are sensitive to methyl methane sulfonate (MMS), X-rays, bleomycin, and hydroxyurea (HU) [8
To provide a comprehensive view of the roles of Dna2p at the replication fork and in other genome maintenance pathways, we conducted a large-scale synthetic lethal screen by synthetic genetic array (SGA) analysis using DNA2
as a query [18
]. Two genes are synthetically lethal if single mutants, defective in either gene, are viable, while double mutants, defective in both genes, are inviable. Two mutants are synthetically sick if the double mutant grows significantly slower than either single mutant. Synthetic lethality is useful for identifying redundancy and complementarity, e.g., pathways that compensate for functional deficiencies in each other or genes encoding products that are both required to efficiently process a common substrate, which is often the case for DNA replication and repair proteins. Synthetic lethal screens not only reveal previously unknown genetic interactions with queried mutants but also how gene products and their corresponding pathways functionally associate.
Our results provide a catalogue of most, if not all, pathways that are interdependent with or require Dna2p, thus revealing both the extent and limits of its multitasking character. The work not only confirms a role in OFP, but also identifies functions in (1) a replication/repair helicase subnet, (2) DSB repair and mismatch repair, (3) the replication stress checkpoint, (4) sister chromatid cohesion, (5) chromatin dynamics, (6) histone modification, and (7) osmotic and oxidative stress responses. In a more general sense, the interactions link a specific network of DNA repair and regulatory pathways to a specific network of replication genes that together maintain high fidelity lagging-strand DNA replication.