Figure 2.

Flp reactions using half-site substrates containing a phosphodiester or methylphosphonate at the scissile position. (A) The methyl substitutions in the R_P and S_P stereoisomers of methylphosphonate are indicated with reference to the achiral phosphodiester. (B) In the schematic representation of a half-site substrate, the horizontal arrows represent the Flp-binding element with its terminal bp abutting the spacer segment shown in bold. The scissile phosphodiester is indicated as ‘P'. Strand cleavage in a half-site traps the tyrosyl intermediate, as the trinucleotide product (5′HO-TTT3′) diffuses away. The 5′ end of the bottom strand is blocked by phosphorylation (denoted by ‘P' in parentheses) from acting as the nucleophile in a joining reaction. (C) A free 5′-OH group on the bottom strand permits the conversion of the tyrosyl intermediate into a hairpin recombinant. Although not shown here, attack by the 5′-OH from a second half-site is also possible. The joined strand would be identical in sequence to the hairpin. (D) Flp-assisted hydrolysis could potentially target the phosphotyrosyl intermediate formed by strand cleavage (top) or the scissile phosphodiester in DNA (bottom).The former activity is called type I endonuclease, the latter, type II.