Crystal Structure of Bacillus DNA Polymerase I Fragment product complex with 11 base pairs of duplex DNA following addition of a dTTP and a dATP residue.

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

Visualizing DNA Replication in a Catalytically Active Bacillus DNA Polymerase Crystal

Crystal structure of a thermostable Bacillus DNA polymerase I large fragment at 2.1 A resolution

Bacillus Fragment (analogous to the E. coli Klenow Fragment)

This protein was isolated from an as yet unnamed novel strain of Bacillus stearothermophilus (see Ref 2).

Ammonium Sulfate, magnesium chloride, MPD, MES, pH 5.8, VAPOR DIFFUSION, HANGING DROP, temperature 290K

1L3S is the same protein bound to the original 9 base pair DNA substrate.

1L3T is a 10 base pair protein-DNA product complex, produced by placing a crystal of the 9 base pair complex (1L3S) in a stabilization solution containing dTTP.

1L3V is a 15 base pair protein-DNA product complex, produced by placing a crystal of the 9 base pair complex (1L3S) in a stabilization solution containing dTTP, dATP, dCTP, and dGTP.

1L5U is a 12 base pair protein-DNA product complex, produced by placing a crystal of the 9 base pair complex (1L3S) in a stabilization solution containing dTTP, dATP, and dCTP.

SEQUENCE
The DNA polymerase gene was cloned from an
organism that was classified as a thermostable
strain of Bacillus stearothermophilus by
ribosomal RNA sequencing. However, this
particular gene has much greater homology with
the analogous gene from Bacillus caldotenax.

The sequence matches residues 297-877 of
SwissProt entry, Q04957, whose source is
Bacillus caldotenax. There are 6 discrepancies
with the Q04957 entry, at residues 456, 505, 512,
550, and 823, as well as a deletion of SwissProt
residue number 576.

oligosaccharide with reducing-end-to-reducing-end glycosidic bond

The Bacillus polymerase was co-crystallized with a DNA containing a 9 base pair duplex and a 6 base 5' template overhang. The crystal was subsequently placed into a stabilizing solution containing dTTP and dATP. The resulting product complex contained 11 base pairs of duplex DNA and a 4 base 5' template overhang. The last three bases in the overhang are disordered and are not included in the model. A single base 3' overhang on the template strand (chain C) assured that the DNA duplex was not bound backwards by the polymerase during crystallization. Electron density was observed for all protein side chains except lysine 4298, which was modelled to the beta carbon. The magnesium at position 920 was assigned due to the low refined b-factor and comparison with a related Klentaq polymerase structure (3KTQ). However, the resolution of the structure prevents a definitive assignment between water and magnesium.

DNA POLYMERASE I (E.C.2.7.7.7)/DNA Complex

Exists as a monomer. One molecule per asymmetric unit.

DNA Polymerase I, DNA Replication, Klenow Fragment, Protein-DNA Complex, TRANSFERASE-DNA COMPLEX