Fig. 3. The structure of the mutant -resolvase
tetramer covalently linked to cleaved DNA. (A) The two site I
DNAs (light and dark green, yellow, and orange coils) are
cleaved into half sites labeled L, R, L', and R'. S10 (blue and
red spheres) in each subunit is covalently linked to the 5'
phosphate of adenine 20 (Ade20, green stick). The subunits of
the resolvase tetramer (blue, green, red, and magenta) can be
divided into two dimers containing antiparallel E helices (L-L'
and R-R') or two site I dimers bound to L-R DNA or L'-R' DNA.
The D and E helices form a four-helix bundle within the
antiparallel dimer. The two antiparallel E helix dimers interact
through a flat interface, and the V114 C114 (21) mutation
in the E helix crosslinks across the flat interface. The
position of the missing phosphates resulting from the use of
symmetrized oligos is marked by an orange sphere; its absence
does not distort the DNA. (B) The active site at the covalent,
cleaved DNA intermediate. A phosphoserine bond formed between
Ade20 and S10. R8, D67, R68, and the two nonbridging oxygens of
the phosphoserine form a hydrogen-bonding network (blue dashed
lines). In the religation step, the free 3'-hydroxyl attacks of
the DNA to be exchanged (black arrow) is presumably in line with
the phosphoserine bond. (C) A view of the tetramer rotated by
90° from the orientation in (A) shows the packing of four E
helices and preceding loops and ß strands.
The above figure is reprinted
by permission from the AAAs: