Formation of ternary HJ-tetrameric Int complexes with arm
oligonucleotides encoding different combinations of P′ sites.
A, HJ-tetrameric Int-arm-type oligonucleotide ternary complexes were
formed by incubating catalytically inactive IntF (Y342F mutation),
32P-radiolabeled Holliday junction, and a 40-bp oligonucleotide
containing either the indicated unsubstituted or biotin dT-substituted
arm-type P′ Int binding sites. The HJ lane lacks protein and arm
oligonucleotide. Complexes were electrophoresed on a 7% polyacrylamide gel and
visualized by autoradiography. B, quantitation of the ratio of arm
oligonucleotides to HJ in the P′1,2,3 and P′1,2,[Bio3] ternary
complexes. 32P signal intensity of P′1,2,3 and
P′1,2,[Bio3] oligonucleotides and fluorescence intensity of a
BODIPY-FL-labeled HJ were measured as described under “Experimental
Procedures” and are reported in arbitrary units (a.u.).
C, DMS protection of P′1,2,3 and P′1,2,[Bio3] 50-bp
oligonucleotides (5′-32P-labeled on the bottom strand)
complexed with HJ-tetrameric Int. After complex formation, the reactions were
treated with DMS, quenched, and electrophoresed on a 7% polyacrylamide gel.
The indicated ternary complexes were excised from the gel, chemically cleaved
at the methylated bases, and analyzed on a sequencing gel. Free P′1,2,3
and P′1,2,[Bio3] oligonucleotides served as the minus protein samples
(0 lanes) to identify diminished (-) and enhanced (+) DNA cleavage
sites. Numbering of the bases refers to the bottom-strand sequence of
attP (52).