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Rec-seq also confirmed earlier findings8 that Cre has a pronounced preference in two areas of loxP: half-site positions 5-7 and 10. We noticed 5.0-fold enrichment of the canonical base at place 10, in keeping with studies that Arg259 participates in hydrogen bonding with the C•G base pair at position 10 (refs. Structural and mutagenesis studies23,26,31 urged that mutation of Arg259 would affect specificity at half-site position 10. Indeed, the Arg259→Ala variant showed a drop in enrichment at position 10 (from 5.0-fold for wild-kind Cre to 1.1-fold for the mutant), with a modest preference for C or T within the left half-site and G or A in the best half-site (Fig. 2b, c). For instance, in helix B, Rec-seq of the Lys43→Ala mutant resulted in a modest drop in specificity relative to wild-sort Cre, while Met44→Ala resulted in greater preference at positions 5 and 10 (Fig. 2d). In helix D, the Lys86→Ala variant showed minimal variations from wild-type Cre (Supplementary Fig. 7a), while the Gln90→Ala variant confirmed general decrease enrichment (Fig. 2e). Within the loop between helices J and K, the Arg282→Ala mutant showed increased, fairly than lower, DNA specificity across loxP (Supplementary Fig. 7b). These outcomes exhibit that Cre’s apparent preference at positions 5-7 results from multiple weak or indirect interactions, fairly than being strongly determined by residues proximal to those positions

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