PDBsum entry 1l3t

Transferase/DNA

PDB id: 1l3t

Contents

Protein chain

580 a.a. *

DNA/RNA

Ligands

GLC-FRU

SO4 ×4

Metals

_MG

Waters ×521

* Residue conservation analysis

References listed in PDB file

Key reference

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

Abstract

DNA polymerases replicate DNA by adding nucleotides to a growing primer strand while avoiding frameshift and point mutations. Here we present a series of up to six successive replication events that were obtained by extension of a primed template directly in a crystal of the thermostable Bacillus DNA polymerase I. The 6-bp extension involves a 20-A translocation of the DNA duplex, representing the largest molecular movement observed in a protein crystal. In addition, we obtained the structure of a "closed" conformation of the enzyme with a bound triphosphate juxtaposed to a template and a dideoxy-terminated primer by constructing a point mutant that destroys a crystal lattice contact stabilizing the wild-type polymerase in an "open" conformation. Together, these observations allow many of the steps involved in DNA replication to be observed in the same enzyme at near atomic detail. The successive replication events observed directly by catalysis in the crystal confirm the general reaction sequence deduced from observations obtained by using several other polymerases and further refine critical aspects of the known reaction mechanism, and also allow us to propose new features that concern the regulated transfer of the template strand between a preinsertion site and an insertion site. We propose that such regulated transfer is an important element in the prevention of frameshift mutations in high-fidelity DNA polymerases. The ability to observe processive, high-fidelity replication directly in a crystal establishes this polymerase as a powerful model system for mechanistic studies in which the structural consequences of mismatches and DNA adducts are observed.

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 #1

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 #2

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