PDBsum entry 1u49

Transferase/DNA

PDB id: 1u49

Contents

Protein chain

580 a.a. *

DNA/RNA

Ligands

GLC-FRU

SO4 ×3

Metals

_MG

Waters ×475

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Error-Prone replication of oxidatively damaged DNA by a high-Fidelity DNA polymerase.

Authors

G.W.Hsu, M.Ober, T.Carell, L.S.Beese.

Ref.

Nature, 2004, 431, 217-221. [DOI no: 10.1038/nature02908]

PubMed id

15322558

Abstract

Aerobic respiration generates reactive oxygen species that can damage guanine residues and lead to the production of 8-oxoguanine (8oxoG), the major mutagenic oxidative lesion in the genome. Oxidative damage is implicated in ageing and cancer, and its prevalence presents a constant challenge to DNA polymerases that ensure accurate transmission of genomic information. When these polymerases encounter 8oxoG, they frequently catalyse misincorporation of adenine in preference to accurate incorporation of cytosine. This results in the propagation of G to T transversions, which are commonly observed somatic mutations associated with human cancers. Here, we present sequential snapshots of a high-fidelity DNA polymerase during both accurate and mutagenic replication of 8oxoG. Comparison of these crystal structures reveals that 8oxoG induces an inversion of the mismatch recognition mechanisms that normally proofread DNA, such that the 8oxoG.adenine mismatch mimics a cognate base pair whereas the 8oxoG.cytosine base pair behaves as a mismatch. These studies reveal a fundamental mechanism of error-prone replication and show how 8oxoG, and DNA lesions in general, can form mismatches that evade polymerase error-detection mechanisms, potentially leading to the stable incorporation of lethal mutations.

Figure 1.
Figure 1: Modes of base pairing for 8oxoG. a, Oxidation of guanine at C8 by reactive oxygen species (ROS). b, 8oxoG in a Watson-Crick base pair with dC. Dashed lines indicate potential hydrogen bonds. c, 8oxoG (syn) in a Hoogsteen base pair with dA (anti).

Figure 3.
Figure 3: Accurate translesion replication of 8oxoG by BF in crystals. a, Schematic of BF active site. During replication, the template base (n, red) moves from the pre-insertion site to the post-insertion site to the DNA duplex binding region. b-d, Structures of accurate 8oxoG replication (blue) are superimposed with structures of unmodified guanine replication (grey). The 8oxoG template base (red) is shown at the pre-insertion site (b) before nucleotide incorporation, the post-insertion site (c) after dCTP incorporation, and the DNA duplex binding region (d) after extension of C 8oxoG.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2004, 431, 217-221) copyright 2004.

Secondary reference #1

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

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 reproduced from the cited reference with permission from Cell Press

Secondary reference #2

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 #3

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