SUPPLEMENTARY INFORMATION
FOR BISWAS, AIHARA, et al.
Purification of -int
-int and -int(75-356) proteins were expressed and purified using a modification of published
methods1,2. In brief, -int proteins were expressed in the E. coli BL21-RIL strain (Invitrogen).
The cells were induced with IPTG and harvested during early log phase to minimize proteolytic
cleavage in the region between the N-domain and CB domain. Cells were lysed by B-PER
reagent (Pierce) in presence of protease inhibitors and 5 mm EDTA. The soluble proteins were
separated by centrifugation and discarded. The pellet from the cell lysate was washed with lysis
buffer containing 2 M NaCl. The insoluble protein was extracted from the pellet in 6 M
guanidium-HCl then renatured by rapid dilution into buffer containing 50 mM Tris, pH 7.5, 1
mM EDTA, 1M NaCl and 10% glycerol. The renatured protein was purified by chromatography
on phosphocellulose, heparin sepharose, Mono-S, and finally a Sephacryl S-100 gel filtration
column to remove small impurities. The purified protein was concentrated to 0.5 mM in 50 mM
Tris (pH 7.0), 0.5 mM EDTA, 1 M NaCl, 2 mM DTT and 10% glycerol prior to storage at 4 °C.
Formation of -int/DNA complexes
-int has a tendency to aggregate in low salt buffers in the absence of DNA. The addition
of core and arm DNAs greatly improves solubility. DNA oligos used for crystallization were gel
purified and annealed in 20 mM Tris (pH 8), 50 mM NaCl, and 0.5 mM EDTA by slow cooling.
For crystallization, -int in 1 M NaCl storage buffer was mixed with core and arm DNAs and
then dialyzed against low salt (50-100 mM NaCl) buffer. An inactive “IntF” mutant (Tyr 342
Phe) of -int was mixed with the Holliday junction and arm DNA (molar ratio 4:1:2.2) and used
for crystallization. The proteins crystallized in ‘post- exchange complex’ harbored the “Int-h”
mutation (Glu 174 Lys), which enhances binding to core sites3,4. -int (Int-h) was mixed with
0.7 molar equivalents of the annealed COC' and arm DNA, then dialyzed against 10mM TrisHCl, pH 7.5, 50mM NaCl, 0.1mM EDTA and 5mM dithiothreitol (DTT) to yield a final protein
concentration of ~0.15mM. For crystallization of the synaptic complex, -int(75-356) was mixed
with the COC' suicide substrate at a molar ratio of 1:0.75, and was dialyzed as above to give the
final protein concentration of ~0.3mM. Crystals of all complexes were grown by hanging drop
vapor diffusion at 21°C by mixing equal volumes of protein-DNA and reservoir solutions. The
crystals of the -int in complex with Holliday junction and arm DNAs grew over 4%
polyethylene glycol (PEG) 8000, 50mM bis-tris propane (pH 6.5), 15mM diammonium
hydrogen phosphate, 5% glycerol, and 5mM DTT. Crystals of -int in complex with COC' and
arm DNAs grew over 12% PEG 4000, 15% isopropanol, 100mM sodium citrate (pH 6.1) and
2mM DTT. -int(75-356) complex crystals grew over a reservoir solution of 30% polyethylene
glycol (PEG) 4000, 50mM sodium citrate (pH 6.6) and 2mM DTT. The space groups, unit cell
dimensions, and x-ray diffraction characteristics of the crystals are shown in Supplementary
Table S1. Crystals were equilibrated with the reservoir solution plus 1, 2-propanediol, glycerol
or ethylene glycol before flash freezing in a cold nitrogen stream.
Structural modeling of the arms of attP
IHF bends DNA by about 160°-180°[REFS 5,6], and the correct spacing of IHF binding sites
in the P and P' arms strongly contributes to recombination activity7. The IHF binding site in the
P' arm is required for both integration and excision (Fig. 1). The P' arm can readily be connected
to the C' branch of core by docking the IHF/DNA crystal structure5 on the -int tetramer (Fig. 5).
2
We define the cleaving strands in the complex as the bottom strands (cleavage sites at C' and B')
according to their orientation relative to the arm DNAs, even though the immobile Holliday
junction (IM3) used for crystallization was designed with the sequence of the top strands
(cleavage sites at C and B) activated for cleavage8. Unlike a native recombination intermediate,
the IM3 Holliday junction cannot isomerize and necessarily binds with the cleavage-active C site
positioned nearest to P'1 arm site -- a position that would be normally occupied by the C' site of
an intact attP DNA.
The DNA bend in the IHF/DNA complex positions the P'1-P'2 binding sites on the Ndomains once a small right-handed twist (~15°) is added to the ends of the DNA extending from
the IHF complex. These are the sites in the P' arm that are bound during excision. A small
delocalized bend of the P' arm is required to correctly position the P'2-P'3 sites used for
integration (Fig. 1a). The P' arm could attain this orientation with the shallower bend angle that
is measured in solution for IHF/DNA complexes6 and by extending the region of the shallow
bend towards the unoccupied P'1 site (Fig. 5).
Interactions of accessory factors with the P arm induce several bends in the DNA that are
less readily predicted. Two IHF binding sites are occupied during integration9, creating a
compound bend that orients P1 over the intasome. In the modeled integrative complex, the IHFmediated bend at the proximal IHF binding site H2 (positioned 32 base pairs from the C binding
site) directs the P arm underneath the tetramer. A second IHF-induced bend at the distal H1 site
reverses the direction of the P arm and brings the P1 site into the vicinity of two unoccupied Ndomains. The P arm segment between two IHF binding sites (H1 and H2) of the integrative
complex can be modeled as a relatively undistorted B-form DNA, although several poly[dA-dT]
tracts in this region may cause an intrinsic curvature. In this orientation, the P arm approaches
3
the N-domain from the same side as the P' arm, though the orientation of P1 makes it lie
antiparallel to the P'2-P'3 sites. This arrangement is in agreement with the antiparallel
orientation of arm DNAs in the crystal structures (Fig. 2) and FRET-based studies10. The P1 site
thus makes specific contacts with one N-domain, whereas the neighboring N-domain could
interact nonspecifically with the DNA immediately distal to P1 (Fig. 5). This may account for
the weaker binding affinity of the P arm compared to that of the P' arm11. During excision, the P
arm adopts a different conformation with the N-domain engaging the P2 site instead of P1. The
P2 site is proximal to and in conflict with the distal IHF binding site (H1) in the P arm (Fig. 1).
A strong bend (> 90°) of the distal P arm is needed in order to bring the P2 site into position.
Two other DNA bending factors required for excision, Xis and/or Fis (the E. coli factor for
inversion stimulation), could form this sharp bend by binding to overlapping sites (between H1
and H2)9,12. The ability of Xis to produce a tight DNA bend has previously been suggested on
the basis of electrophoretic mobility studies13. Considering together the positions of the Xis, Fis,
and P2 binding sites along with the overall shape of the tetramer, it is likely that the P arm is
sharply bent and in very different conformations during excision and integration. Both modeled
conformations of the P arm are compatible with existing data, though other conformations may
occur during the course of either recombination reaction.
_____________________________________________________________________________
1.
2.
3.
Tirumalai, R. S., Kwon, H. J., Cardente, E. H., Ellenberger, T. & Landy, A. Recognition
of core-type DNA sites by lambda integrase. J Mol Biol 279, 513-27 (1998).
Aihara, H., Kwon, H. J., Nunes-Duby, S. E., Landy, A. & Ellenberger, T. A
conformational switch controls the DNA cleavage activity of lambda integrase. Mol Cell
12, 187-98 (2003).
Lange-Gustafson, B. J. & Nash, H. A. Purification and properties of Int-h, a variant
protein involved in site-specific recombination of bacteriophage lambda. J Biol Chem
259, 12724-32 (1984).
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4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Patsey, R. L. & Bruist, M. F. Characterization of the interaction between the lambda
intasome and attB. J Mol Biol 252, 47-58 (1995).
Rice, P. A., Yang, S., Mizuuchi, K. & Nash, H. A. Crystal structure of an IHF-DNA
complex: a protein-induced DNA U-turn. Cell 87, 1295-306 (1996).
Swinger, K. K. & Rice, P. A. IHF and HU: flexible architects of bent DNA. Curr Opin
Struct Biol 14, 28-35 (2004).
Thompson, J. F., Snyder, U. K. & Landy, A. Helical-repeat dependence of integrative
recombination of bacteriophage lambda: role of the P1 and H1 protein binding sites. Proc
Natl Acad Sci U S A 85, 6323-7 (1988).
Azaro, M. A. & Landy, A. The isomeric preference of Holliday junctions influences
resolution bias by lambda integrase. Embo J 16, 3744-55 (1997).
Landy, A. Dynamic, structural, and regulatory aspects of lambda site-specific
recombination. Annu Rev Biochem 58, 913-49 (1989).
Radman-Livaja, M. et al. Arm sequences contribute to the architecture and catalytic
function of a lambda integrase-Holliday junction complex. Mol Cell 11, 783-94 (2003).
Sarkar, D. et al. Differential Affinity and Cooperativity Functions of the Amino-terminal
70 Residues of lambda Integrase. J Mol Biol 324, 775-89 (2002).
Nash, H. A. in Escherichia coli and Salmonell typhimurium: Cellular and Molecular
Biology (ed. F.C. Neidhardt, R. C., J.L. Ingraham, E.C. Lin, K.B. Low, et al.) 2263-2376
(ASM Press, Washington D.C., 1996).
Thompson, J. F. & Landy, A. Empirical estimation of protein-induced DNA bending
angles: applications to lambda site-specific recombination complexes. Nucleic Acids Res
16, 9687-705 (1988).
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