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Figure 1.
Figure 1. Crystal Structure of Dbh Extending DNA Synthesis
beyond a Single-Base Deletion in a Repetitive Sequence
(A)
Ternary complex structure. The polymerase and primer-template
DNA are shown in ribbons representation, the incoming dNTP is
shown as a ball-and-stick model, and the Ca^2+ ion (bound in the
metal B position) is shown as a sphere (green). Residues Arg333
and Tyr249 that interact with the bulged template cytosine at
position −3 are shown in ball-and-stick representation.
Coloring is as follows: palm (magenta), thumb (green), fingers
(blue), C-terminal domain (orange), linker between polymerase
and C-terminal domains (pale yellow); and DNA (white). The
template strand single-base deletion hot spot sequence
(5′-GCCC-3′) is labeled and highlighted in red.
(B)
Protein-DNA interactions. Hydrogen bonds and van der Waals
contacts are indicated by dotted lines; residues that contact
the DNA with side-chain atoms are labeled in boldface; other
residues that contact main-chain atoms are labeled in plain
type. Residues are colored according to domain, as in (A).
Nucleotides are numbered relative to the templating base
(position 0). The complete sequence of the DNA oligonucleotides
used is shown; the template-strand hot spot sequence is
highlighted in red. Unpaired nucleotides at the 5′ and 3′
ends of the template strand (positions +2 and −12) were not
clearly visible in the electron density maps and were thus not
modeled. Residues in the flexible loop that are close enough to
contact the backbone of the template in positions −1, −2,
and −3 are boxed and labeled in italic font; precise contacts
are not identified, because of the weak electron density in this
part of the protein. The Ca^2+ ion bound at the active site in
the metal B position is shown as a circle labeled B.
(C–E) The active sites and primer-template DNA sequences are
shown for (C) preinsertion binary complex, (D) insertion ternary
complex, and (E) postinsertion binary complex. For each
structure, a final refined 2F[o] − F[c] electron density map,
calculated using the same resolution limits (Table 1) as used
for refining each structure and contoured at 1.2 σ (gray mesh),
is shown for the DNA substrates, calcium, and Dbh residues Asp7,
Asp105, Glu106, Tyr10, Tyr48, and Arg51 and is superimposed on
the final refined model. The incoming nucleotide (ddGTP), DNA,
and selected residues in the active site pocket are shown in
stick representation; other portions of the protein are shown in
ribbons representation. The 3′ primer terminus (dG) of each
structure is labeled. Atoms are colored by element: oxygen
(red), nitrogen (blue), phosphate (yellow), carbon (white), and
calcium (green). Coordination of the Ca^2+ ion in the ternary
complex is shown with dotted lines (dark gray).
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