Arm-type site protection patterns in the excisive recombination HJ
intermediate. Excisive recombination reactions between attL and
attR were carried out in the presence of the HJ-trapping peptide,
treated with DNase I, and deproteinated as described under “Experimental
Procedures.” Selectively radiolabeled DNase I fragments were
fractionated on a sequencing gel and visualized by autoradiography.
A, a 5′-32P-labeled primer complementary to the top
strand of the P′ arm was extended into the P arm of the HJ to label and
visualize the cleavage patterns of the top strand of the HJ in this region.
The predicted location of the putative non-canonical site lies within the H1
site and is denoted by the NC label and a dashed line. B,
the P′ arm of the HJ complex was radiolabeled using a
5′-32P-labeled primer for the top strand of the attR
substrate. The P′ arm fragments of the HJ migrate in the upper portion
of the sequencing gel, above the smaller substrate attR fragments,
which migrate just below the H′ site (partially visible in gel). In both
A and B a small linear attP fragment provides the
protein-free DNA control for the footprints (0 lanes). Dotted
lines mark regions of significantly diminished (-) or enhanced (+)
cleavage.