Thesis (Ph. D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2007.
During eukaryotic DNA replication, proteins function coordinately, replicating the genome accurately and maintaining genome integrity. Due to the antiparallel nature of DNA, leading strand replication proceeds continuously, while lagging strand replication occurs discontinuously via Okazaki fragment maturation. Okazaki fragments are nucleotide segments initiated by an RNA/DNA primer, approximately 20-30 nucleotides in length. Generation of continuous doublestranded DNA through joining of the fragments requires removal of the RNA/DNA primer. DNA polymerase δ extends the upstream Okazaki fragment, displacing the 5′ end of the downstream primer into a single-stranded flap, which is removed by nuclease cleavage. One pathway for primer removal involves repeated polymerase δ displacement and flap endonuclease 1 (FEN1) cleavage of short flaps. A second pathway involves long flap displacement by polymerase δ, coating of long flaps by the single-stranded DNA binding protein, replication protein A (RPA), and sequential cleavage by Dna2 nuclease and FEN1. To establish the predominant pathway for primer removal, flap displacement and FEN1 cleavage were measured in a reconstituted system in vitro utilizing purified proteins from Saccharomyces cerevisiae (S. cerevisiae) and oligonucleotide substrates, which represent Okazaki fragment intermediates. Although results demonstrate that some flaps escape cleavage and grow to approximately 20-30 viii nucleotides in length, the majority of flaps are cleaved while short by FEN1. This suggests that FEN1 is sufficient to remove the RNA/DNA primer through repeated cleavage of short flaps. However, some flaps become long enough to bind RPA and require cleavage by the two-nuclease pathway. Addition of RPA into the system reveals that RPA only moderately inhibits FEN1 long flap cleavage. However, this inhibition is promoted by Pif1 addition. Pif1 is a 5′-3′ helicase, which interacts genetically with Dna2 in S. cerevisiae. Lethality of dna2Δ is suppressed by pif1Δ, implying that Pif1 creates a need for Dna2. Furthermore, in the absence of RPA, Pif1 improves flap cleavage by FEN1 in vitro. Together, the data suggest that Pif1 accelerates flap growth, generating more flaps that allow RPA binding, blocking FEN1 tracking to its cleavage site. Therefore, in Okazaki fragment maturation, Pif1 likely directs flaps toward the two-nuclease pathway, requiring Dna2 cleavage for primer removal.
