Extended Data Figure 6. Hybrid intasomes: structure-based design and validation in vitro.

a, Views on the environment of Lys120 and Asp273 PFV IN residues within the intasome structure. Protein is shown as cartoons with side chains of selected amino acid residues shown as sticks; the cartoons and carbon atoms of the inner and outer IN chains are shown in green and light-orange, respectively. Lys120 of the outer and Asp273 of the inner IN subunit are involved in a network of interactions; in contrast, Lys120 of the inner and Asp273 of the outer IN subunit are solvent-exposed. Consequently, IN mutants harboring substitutions of Lys120 or Asp273 can only play a role in the inner or outer intasomal subunits, respectively; b, PFV IN mutants K120E and D273K are co-dependent for intasome assembly. Products of intasome assembly using WT, D273K, K120E PFV IN or an equimolar mixture of D273K and K120E INs were separated by size exclusion chromatography. Elution positions of the intasome, IN and free DNA are indicated. The assembly was successful with WT IN or with a mixture of the two IN mutants, but not with either of the IN variants separately; c, Validation of the hybrid intasome design. Left: possible types of strand transfer products obtained by reacting the intasome with circular DNA target (pGEM, black lines). Full-site integration (strand transfer involving both intasomal DNAs, dark blue lines) results in a linear concerted product, which may be targeted by further strand transfer events. Half-site integration (strand transfer involving a single intasomal DNA end) results in a circular branched single-end product. Right: strand transfer assays using mutant intasomes and circular pGEM DNA target. The intasomes were assembled using WT IN or a mixture of K120E and D273K mutants, as indicated atop the gel. IN variants indicated with a cross (†) additionally incorporated the E221Q amino acid substitution that disables the enzyme active site. Reaction products were separated by agarose gel electrophoresis. Intasomes were used at indicated concentrations; the leftmost lane contained a mock sample, which received no intasome. Migration positions of the reaction products, intasomal DNA and unreacted supercoiled (s.c.) pGEM are indicated to the right of the gel. As predicted, the strand transfer function of the hybrid intasome strictly requires the active site from the K120E (inner) IN subunit, but not the D273K (outer) subunit; d, Strand transfer activity of mutant intasomes on naked plasmid DNA. Mutations indicated in orange or green were restricted to the outer or inner subunits of the hybrid PFV intasome, respectively.