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Figure 6.
Fig. 6. Proposed CPT binding mode. (A) A schematic
representation of the key hydrogen bond and ring-stacking
interactions made between the human topo I-DNA covalent complex
and CPT in the proposed CPT binding mode. The atomic
nomenclature for CPT is also indicated. (B) Stereoview of the
proposed binding mode of CPT to the covalent human topo I DNA
complex. The active^ lactone form of CPT (20-S-camptothecin, in
green) is shown stacked^ between the terminal +1 guanine
nucleotide from the cleaved strand^ (+1 Gua, in yellow, which is
reoriented from the observed position as described below), and
the side chain of Asn722, which provides interactions with the
A-ring of CPT (the cleaved^ strand is rendered in light and dark
magenta upstream and downstream of the cleavage site,
respectively). The carbonyl oxygen at the^ 17 position in CPT
makes a hydrogen bond with the NH[2] group on the pyrimidine
ring of the +1 cytosine. The side chains of active-site^
residues Tyr723, Arg488, and Arg590 are shown in cyan. The side
chain residues that, if singly mutated, result in a
CPT-resistant phenotype [Phe^361, Gly363, and Arg364 of region 1
(see text); Asp533 and Asn722 of region 2] are shown in tan. The
side chain conformations of^ Arg364 and Asp533 have been altered
slightly from the final structure of the covalent complex to
allow for optimal hydrogen bonding to the double-bonded^ lactone
oxygen and the hydroxyl at the 20-S chiral center of CPT,
respectively. Modifications to the 10 and 11 positions of CPT
may require some minor shifts in the positions of residues
Lys720 and Leu721 of topo I, which exhibit relatively high
temperature factors (for example, 55 to 65 Å2) in the
structure of the covalent complex. The proposed conformation of
the +1 Gua nucleotide was inspired by flipped-out bases
observed^ experimentally by Sussman and co-workers (62), but was
further optimized by rotations about bonds in the intact
phosphate between the +1 and +2 nucleotides. Because this base
is now a terminal nucleotide in the cleaved strand, it is less
contrained by the^ ribose-phosphate backbone and is more free to
rotate to positions outside the DNA duplex.
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