Characterization of the interaction of Saccharomyces cerevisiae strand exchange protein 1 with DNA.
We have analyzed in greater detail the interaction of strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae with DNA. The binding site size of Sep1 on single-stranded DNA (ssDNA) was determined to be 70 nucleotides per protein monomer using a fluorescence assay and 100 nucleotides using an exonuclease titration technique. The amount of Sep1 required for maximum aggregation of ssDNA was the amount needed to saturate the DNA. When double-stranded DNA (dsDNA) and ssDNA were both present, the duplex DNA was efficiently aggregated only at protein concentrations above that required for saturation of ssDNA. Strand exchange reactions with blunt-ended linear dsDNA and homologous ssDNA substrates required saturation of the ssDNA with Sep1 since free Sep1 is needed for exonuclease activity to initiate pairing with the dsDNA substrate. Preincubation of Sep1 with resected duplex DNA before adding ssDNA allowed joint molecule formation to occur at protein concentrations at least 10-fold below that required for saturation of the ssDNA. However, preincubation of Sep1 with ssDNA before the addition of resected duplex DNA required saturating amounts of Sep1 for joint molecule formation to occur. These results suggest that pairing requires Sep1 on both the ssDNA and the resected ends of the dsDNA.
Johnon
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Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
Kolodner
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RD
D001483:Base Sequence; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism; D005095:Exoribonucleases; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D005092:Exonucleases; D005095:Exoribonucleases; C068398:XRN1 protein, S cerevisiae