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Repair of dsDNA breaks requires processing to produce 3'-terminated ssDNA. We biochemically reconstituted DNA end resection using purified human proteins: Bloom helicase (BLM); DNA2 helicase/nuclease; Exonuclease 1 (EXO1); the complex comprising MRE11, RAD50, and NBS1 (MRN); and Replication protein A (RPA). Resection occurs via two routes. In one, BLM and(More)
The precise machineries required for two aspects of eukaryotic DNA replication, Okazaki fragment processing (OFP) and telomere maintenance, are poorly understood. In this work, we present evidence that Saccharomyces cerevisiae Pif1 helicase plays a wider role in DNA replication than previously appreciated and that it likely functions in conjunction with(More)
The Mre11/Rad50/Xrs2 complex initiates IR repair by binding to the end of a double-strand break, resulting in 5' to 3' exonuclease degradation creating a single-stranded 3' overhang competent for strand invasion into the unbroken chromosome. The nuclease(s) involved are not well understood. Mre11 encodes a nuclease, but it has 3' to 5', rather than 5' to 3'(More)
Yeast Dna2 helicase/nuclease is essential for DNA replication and assists FEN1 nuclease in processing a subset of Okazaki fragments that have long single-stranded 5' flaps. It is also involved in the maintenance of telomeres. DNA2 is a gene conserved in eukaryotes, and a putative human ortholog of yeast DNA2 (ScDNA2) has been identified. Little is known(More)
The repair of DNA double-strand breaks (DSBs) by homologous recombination requires processing of broken ends. For repair to start, the DSB must first be resected to generate a 3'-single-stranded DNA (ssDNA) overhang, which becomes a substrate for the DNA strand exchange protein, Rad51 (ref. 1). Genetic studies have implicated a multitude of proteins in the(More)
In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap(More)
The replication initiation protein Cdc6p forms a tight complex with Cdc28p, specifically with forms of the kinase that are competent to promote replication initiation. We now show that potential sites of Cdc28 phosphorylation in Cdc6p are required for the regulated destruction of Cdc6p that has been shown to occur during the Saccharomyces cerevisiae cell(More)
We have recently described a new helicase, the Dna2 helicase, that is essential for yeast DNA replication. We now show that the yeast FEN-1 (yFEN-1) nuclease interacts genetically and biochemically with Dna2 helicase. FEN-1 is implicated in DNA replication and repair in yeast, and the mammalian homolog of yFEN-1 (DNase IV, FEN-1, or MF1) participates in(More)
SUMMARY The polyguanine-rich DNA sequences commonly found at telomeres and in rDNA arrays have been shown to assemble into structures known as G-quadruplexes, or G4 DNA, stabilized by base-stacked G quartets, an arrangement of four hydrogen bonded guanines. G4 DNA structures are resistant to the many helicases and nucleases that process intermediates(More)
To help define the pathologies associated with yeast cells as they age, we analyzed the transcriptome of young and old cells isolated by elutriation, which allows isolation of biochemical quantities of old cells much further advanced in their life span than old cells prepared by the biotin-streptavidin method. Both 18-generation-old wild-type yeast and(More)