λ site-specific recombination and models of arm-type Int binding
in integrative and excisive recombination HJ intermediates. A,
integration requires Int and IHF and proceeds via Int-catalyzed top-strand
cleavages (small arrows) at core sites C and B followed by strand
swapping at the left boundary of the 7-bp homologous overlap region (top
strands in red, bottom strands in green) to form the
integrative HJ intermediate. After isomerization, the HJ is resolved by
bottom-strand cleavages at core sites C′ and B′ to generate the
attR and attL of the integrated prophage. Excision requires
Int, IHF, Xis, and FIS (when Xis is limiting) and proceeds with the same order
of strand exchanges (top strands first) to regenerate attP and
attB. Protein binding sites required for each reaction are
highlighted in bold: Int arm-type sites (P1, P2, P′1,
P′2, and P′3), Int core-type sites (C, B, C′, and B′),
IHF binding sites (H1, H2, and H′), Xis binding sites (X1, X1.5, and
X2), and FIS binding site (F). Structure-predicted, putative non-canonical Int
binding sites (12) are marked
NC with a dashed line over their predicted locations in
attP and attR; half-arrows above the arm-type sites
denote their relative polarity. B, C, and D are schematic
depictions of tetrameric Int-HJ structure and the predicted arm-type site
binding patterns in the modeled recombination intermediates (adapted from
Biswas et al. (12)).
B, in the x-ray crystal structure of the minimized, symmetrized
tetrameric Int-HJ complex, four core-type Int sites (C, B, C′, and
B′) comprising the four branches of the HJ (dotted lines) are
bound by the Int core binding and catalytic domains (large orange and
green circles), each of which is joined to its corresponding
amino-terminal domain (small circles) by a linker region (small
lines). The amino-terminal (N) Int domains are circularly
displaced and bind to the indicated arm-type site, P′1, or P′2
encoded on two short oligonucleotides (gray bars). C, in the
modeled integrative recombination intermediate, the P′1 and P′2
sites present in the crystal structure are replaced by arm-type sites P1,
P′2, P′3, and a non-canonical site (NC, bold).
D, in the modeled excisive recombination intermediate the P′1
and P′2 sites present in the crystal structure are replaced by arm-type
sites P2, P′1, P′2, and a non-canonical site (NC, bold).
In panels C and D, the solid heavy arrows denote
where the arm-type site DNA would connect (in cis) to branches of the
HJ (see depictions of HJ intermediates in panel A, above); red
arrows depict connectivity between the P arm DNA and the HJ branch
containing the C core-type site; black arrows depict connectivity
between the P′ arm DNA and the HJ branch containing the C′
core-type site; the regions of DNA not depicted in these schematics encode the
binding sites for the accessory proteins (see panel A above) that
facilitate the formation of Int bridges between the arm- and core-type sites
by bending the intervening DNA.