Fidelity of copying of the genome during DNA replication is maintained at a robust level by a poorly understood network of intersecting pathways. Specific mechanisms by which these pathways protect the genome remain uncharacterized due to the complexity of the underlying processes at the replication fork and their regulation. A major challenge is to understand if and how the replication apparatus coordinates the genome maintenance machineries. Recently, we have used global genetic interaction screens (SGA) and have defined an elaborate network of replication, repair, and regulatory (checkpoint and cell cycle) genes that we propose preserves the integrity of the lagging strand at the replication fork. DNA polymerase delta, FEN1 nuclease, and the essential replication helicase/nuclease Dna2 are key hubs in this network of Okazaki fragment synthesis and processing (OFP) enzymes, as are the Sgs1, Rrm3, Pif1, and Srs2 helicases. The pathways in the network define major avenues for guarding the genome and have implications for understanding of diseases such as cancer and aging that may derive from genome instability. Two major specific insights have been gained from analysis of the network and dictate our new directions: (1) We have found that the requirement for Dna2 protein for viability in yeast can be bypassed by deletion of another helicase, Pif1. Genetic evidence further suggests strong interaction of both Dna2 and Pif1 with DNA polymerase delta. We will probe the contribution of Pif1 to accurate lagging strand replication, using biochemical reconstitution, emphasizing the contribution of Pif1 to the well-characterized reactions of Dna2, FEN1, and pol delta on model substrates mimicking OFP intermediates. (2) We have found that deletion of DNA2 suppresses the excessive telomere elongation observed in pif1 mutants, adding to significant previous evidence that Dna2 functions at telomeres. We will study lagging strand synthesis on telomeric DNAs as well as interaction of Dna2 with various G-quadruplex Structures that may occur at telomeres (and elsewhere in the genome). Additional possible roles for Dna2 at telomeres will be tested, such as a role in recruiting telomerase and in degrading uncapped telomeres. Telomere length in dna2 mutants will be examined and compared with length in other mutants affecting telomere homeostasis.
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