Arm-type site and minimal DNA requirements in excisive
recombination. A, biotin interference assay of excisive
recombination. att sites containing a single biotin dT substitution
at one of the indicated positions (t.s., top strand, b.s.,
bottom strand) were recombined with unsubstituted partners, and the
recombination efficiency was normalized to that of a control reaction with no
substitutions (20–35% recombination efficiency). Based on the x-ray
crystal structure (12), the
biotin at -117 (*) is predicted to interfere with the putative
non-canonical Int binding in the same way that biotin at position -107 blocks
Int binding at P2. Biotin dT substitutions at positions -122 and -112 required
changing the wild-type sequence from a G and a C, respectively; in each case
the unsubstituted control attR was also mutated to T. Numbering of
the att bases corresponds to the sequence of attP
(14,
52). B, resection
analysis of excisive recombination. Full-length attR substrate
(wild-type(WT)) and substrates with the putative non-canonical site
resected (NC resect) and P2 resected (P2 resect) were
recombined with an attL lacking P′3(P′3 resect), which is
not required for excisive recombination (see “Results”). Bent
arrows beneath the attR and attL sequences in
A denote resection endpoints and the direction of remaining sequence
in the attR and attL substrates. The P2 resect substrate was
generated by an NdeI digest, and the P′3 resect substrate contained a
CGC insertion after base +74 to stabilize the resected end. Wild-type
attR substrate contained a mutation in the H1 site to correct for the
absence of the H1 site in the NC resect and P2 resect substrates. IHF binding
at the H1 site inhibits excisive recombination and, thus, mutating or removing
this site is stimulatory
(16).