PDBsum entry 1nke

Transferase/DNA

PDB id: 1nke

Contents

Protein chain

580 a.a. *

DNA/RNA

Ligands

GLC-FRU ×2

DCP

SO4 ×5

Metals

_MG

Waters ×441

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structures of mismatch replication errors observed in a DNA polymerase.

Authors

S.J.Johnson, L.S.Beese.

Ref.

Cell, 2004, 116, 803-816. [DOI no: 10.1016/S0092-8674(04)00252-1]

PubMed id

15035983

Abstract

Accurate DNA replication is essential for genomic stability. One mechanism by which high-fidelity DNA polymerases maintain replication accuracy involves stalling of the polymerase in response to covalent incorporation of mismatched base pairs, thereby favoring subsequent mismatch excision. Some polymerases retain a "short-term memory" of replication errors, responding to mismatches up to four base pairs in from the primer terminus. Here we a present a structural characterization of all 12 possible mismatches captured at the growing primer terminus in the active site of a polymerase. Our observations suggest four mechanisms that lead to mismatch-induced stalling of the polymerase. Furthermore, we have observed the effects of extending a mismatch up to six base pairs from the primer terminus and find that long-range distortions in the DNA transmit the presence of the mismatch back to the enzyme active site, suggesting the structural basis for the short-term memory of replication errors.

Figure 2.
Figure 2. DNA Mismatches Bound at the Polymerase Postinsertion SiteThe bases are shown in the same orientation and location as the G•C base pair in Figure 1B. Left, hydrogen bonding pattern. Right, superimposition of the molecular surface of the mismatch (red) and cognate G•C base pair (yellow, PDB ID 1L3S) bound at the postinsertion site, highlighting differences in shape and location of the primer terminus.

Figure 4.
Figure 4. Extension of a G•T Mismatch by Successive Rounds of ReplicationThe conformation of the G•T mismatch is shown at each position (left), including interacting water molecules (red spheres). Dashed lines indicate potential hydrogen bonds. At the n-3 and n-4 positions, hydrogen bonds are shown between groups within the appropriate distance (≤3.2 Å) and correspond to tautomerization or ionization of one of the bases (see text). A schematic representation (right) of the mismatch complex, drawn and color coded as described in Figure 1, Figure 2 and Figure 3, indicates regions of the polymerase active site that are disrupted upon binding of the mismatch (red line). Mismatch binding at positions n-1 to n-4 along the DNA duplex binding region (gray) results in a distorted open conformation at the polymerase active site as described by mechanism 1 (Figure 3). The normal open conformation observed with homoduplexes is fully restored when the mismatch is bound at the n-6 position.

The above figures are reprinted by permission from Cell Press: Cell (2004, 116, 803-816) copyright 2004.

Secondary reference #1

Title

Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations.

Authors

S.J.Johnson, J.S.Taylor, L.S.Beese.

Ref.

Proc Natl Acad Sci U S A, 2003, 100, 3895-3900. [DOI no: 10.1073/pnas.0630532100]

PubMed id

12649320

Figure 2.
Fig. 2. Active site superposition of open and closed BF structures. (A) The 11-bp open binary complex (yellow) and the closed ternary complex (blue) are shown in stereo view. The largest conformational differences occur in the fingers domain, including the O helix, O1 helix, and preinsertion site. The acceptor template base (n) occupies the preinsertion site in the open conformation and the insertion site in the closed conformation. (B) A close-up view of the preinsertion site. The locations of the conserved Tyr-714 are indicated.

Figure 3.
Fig. 3. Conformational interlocks during DNA synthesis. A schematic overview of the polymerase active site (A) and atomic coordinates (B) derived from the open and closed BF structures represent a complete round of DNA synthesis. The conformational changes described here are presented in animated form in Movie 1, which is published as supporting information on the PNAS web site. The reaction cycle starts with the acceptor template base (n, red) bound at the template preinsertion site (between the O and O1 helices; green shading); Tyr-714 blocks access to the insertion site (blue shading) and stacks with the n-1 base pair at the postinsertion site (gray shading). Formation of the closed conformation involves rearrangement of the O and O1 helices, which simultaneously blocks the template preinsertion site and unblocks the insertion site. These rearrangements move the acceptor template base (n) to the insertion site, where it pairs with an incoming dNTP (green). Nucleotide incorporation occurs on formation of a cognate base pair and proper assembly of the catalytic site (orange shading). The cycle is completed with translocation of the DNA by one base pair position. The polymerase resets to the open conformation in preparation for the next round of DNA synthesis.

Secondary reference #2

Title

Visualizing DNA replication in a catalytically active bacillus DNA polymerase crystal.

Authors

J.R.Kiefer, C.Mao, J.C.Braman, L.S.Beese.

Ref.

Nature, 1998, 391, 304-307. [DOI no: 10.1038/34693]

PubMed id

9440698

Figure 1.
Figure 1 Structure of the Bacillus fragment with duplex DNA bound at the polymerase active site. The Bacillus fragment molecular surface is coloured according to its proximity to the DNA, with all points less than 3.5 ? coloured magenta, between 3.5 and 5.0 ? yellow, and greater than 5 ? blue. Bound water molecules were not included in this calculation.

Figure 4.
Figure 4 Polymerase active site with observed DNA and modelled dTTP. The position of dTTP (violet) was based on the -polymerase complex18, adjusted such that the base ring stacks with the primer and one oxygen from each phosphate group was within 3 ? of the observed metal ion (gold). The sugar pucker of the primer terminus was made C3'-endo, which shifted its 3'-OH to within 1.7 ? of the modelled -phosphate of the dTTP. A second metal ion (violet) was modelled to be within 3 ? of the 3'-OH of the primer, the -phosphate group, and residues Asp 830 and Glu 831. The observed 5' template overhang cannot accept an incoming dNTP without a conformational change of the O helix.

The above figures are reproduced from the cited reference with permission from Macmillan Publishers Ltd

Secondary reference #3

Title

Crystal structure of a thermostable bacillus DNA polymerase i large fragment at 2.1 a resolution.

Authors

J.R.Kiefer, C.Mao, C.J.Hansen, S.L.Basehore, H.H.Hogrefe, J.C.Braman, L.S.Beese.

Ref.

Structure, 1997, 5, 95. [DOI no: 10.1016/S0969-2126(97)00169-X]

PubMed id

9016716

Figure 3.
Figure 3. Comparison of 3'-5' exonuclease active sites. Stereo diagram of the BF polymerase vestigial exonuclease active site (red) with the position of a portion of the structure of the KF active site (gold) [4] superimposed. The KF Ca backbone schematic is accompanied by is two bound zinc atoms (green), and three nucleotides (black) from the KF editing complex [11]. The KF residues shown (yellow) are the four residues that bind the two metal ions essential for catalysis. These essential KF sidechains Asp355, Glu357, Asp424, and Asp501 correspond to BF residues Val319, Glu321, Ala376, and Lys450, respectively (shown in blue). Also shown in blue are two BF proline residues (438 and 441) that may be responsible for the collapse of a loop between helices E[1] and F (dotted line) into the exonuclease cleft not observed in KF. (Drawn with RIBBONS [71].)

The above figure is reproduced from the cited reference with permission from Cell Press