FIG 7.

Helix E residues controlling synaptic complex formation. (A) Sequence of the N-terminal segment of the E helices of SRs over the region in contact with their catalytic domain or partner helix E. Blue-shaded residues are the positions of gain-of-function mutations in A118 integrase (this work), Hin DNA invertase (38, 39, 43), Tn3 or γδ resolvase (37, 41), Sin resolvase (42), and ϕC31 Int (61) that enhance SC formation. Three LSR sequences are also given with gray shading, indicating identities with the A118 integrase: a putative conjugative transposon recombinase from Clostridium difficile used for structural modeling (PDB accession number 3G13) (see panel H), the TP901 integrase, and the more distantly related ϕC31 integrase. (B) SC formation by A118 mutants. Reactions with the different A118 proteins employed 100-bp ³²P-labeled attB and excess 60-bp attP fragments. Proteins in the right panel have their CC motifs deleted. (C) Migration of SCs formed by Int-M117I and -L127V with 100-bp ³²P-labeled attB and excess 60- or 200-bp attP fragments. (D) Specificity of att site synapsis by A118 mutants. Example gels are given for Int-M117I and -L121I reactions with 100-bp ³²P-labeled attB and excess 60-bp attB, attP, attL, and attR fragments in the absence and presence of Gp44. Note that Gp44 binding (*) to the Int dimer on attB shifts the complex to a faster-migrating species. A complete set of specificity gels is provided in Fig. S5 in the supplemental material. (E) Summary of att site synapsis specificities by the A118 integrase mutants. +^f indicates a higher-mobility complex that is missing the attP DNA. (−) indicates not detected but that a higher-mobility SC could be obscured by the Gp44-bound dimeric complex (but see Fig. S5B). (F) SEC of Int-M117 without and with incubation with attP DNA. The trace of the DNA only (60-bp attP) and peaks of MW standards (catalase, 232 kDa; aldolase, 158 kDa; BSA, 67 kDa) are shown. AU, arbitrary units. (G) Recombination frequencies of wt and mutant A118 integrases with or without their CC motifs. Results are the averages and standard deviations from 20-min attB × attP deletion reactions relative to the wt (0.556 ± 0.033 deletions/substrate molecule). (H) Structural model of the A118 integrase dimer over the catalytic domain and helix E regions highlighting the locations of the SC-forming mutations in helix E. The catalytic domain of one subunit is rendered as a surface with its helix E as a dark green ribbon and the native side chains of the mutant residues in red (C_α red sphere for Gly119). The model is based on the structure under PDB accession number 3G13 (see Materials and Methods).