Abstract
Contact points between the FLP protein of the yeast 2-micron plasmid and its recombination site have been defined. Important features of the region previously defined as the minimal recombination site in vitro include a pair of 13-base pair inverted repeats separated by an 8-base pair spacer. The two FLP protein-binding sites within this region are 12 base pairs in length. In each case they include the internal 11 base pairs of one of the 13-base pair repeats, as well as the adjacent base pair within the spacer. The internal 6 base pairs within the spacer are not involved in binding or recognition by FLP protein. When the size of the spacer is increased or decreased by one base pair, the distance between the cleavage points is also increased or decreased correspondingly by one base pair. Points of cleavage are unaffected by changes in the spacer sequence. Specific contact points involving purine residues, identified by methylation protection and recombination interference experiments, are located in both the major and minor grooves of the DNA. Additional contact points between FLP protein and phosphate groups in the phosphate-deoxyribose backbone are clustered near the cleavage sites.
Highlights
Data at position 6 was obscured by a band resulting six base pairsof the spacer, consistenwt ith previous evidence from FLP-mediated cleavage of the substrate DNA and no that these positions are not recognized by FLP protein
Methylation of any of the guanine residues withinthe 10 base pairs of each repeat immediately flanking the spacercaused a pronounced inhibition of the recombination reaction
These results indicate that prominent interactions of FLP protein with its recombination siteoccur in both the major and minor grooves
Summary
Samples were precipitated with butanol, resuspended in 150 pI 1% sodium dodecyl sulfate, reprecipitatedwith butanol, washed with 95%. Samples were resuspended in 2 p1 of buffer X. Additional samples of the same labeled DNA fragment were subjected to the Maxam-GilbertG reaction (14). All samples were electrophoresed on the sampeolyacrylamide gel under denaturing conditions as described above
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