Summary information and primary citation
- PDB-id
-
4flz;
DSSR-derived features in text and
JSON formats
- Class
- transferase-DNA
- Method
- X-ray (3.2 Å)
- Summary
- Pyrococcus abyssi b family DNA polymerase bound to a
dsDNA, in edition mode
- Reference
-
Gouge J, Ralec C, Henneke G, Delarue M (2012): "Molecular
Recognition of Canonical and Deaminated Bases by P.
abyssi Family B DNA Polymerase."
J.Mol.Biol., 423, 315-336. doi:
10.1016/j.jmb.2012.07.025.
- Abstract
- Euryarchaeal polymerase B can recognize deaminated
bases on the template strand, effectively stalling the
replication fork 4nt downstream the modified base. Using
Pyrococcus abyssi DNA B family polymerase (PabPolB), we
investigated the discrimination between deaminated and
natural nucleotide(s) by primer extension assays,
electrophoretic mobility shift assays, and X-ray
crystallography. Structures of complexes between the
protein and DNA duplexes with either a dU or a dH in
position +4 were solved at 2.3Å and 2.9Å resolution,
respectively. The PabPolB is found in the editing mode. A
new metal binding site has been uncovered below the
base-checking cavity where the +4 base is flipped out; it
is fully hydrated in an octahedral fashion and helps guide
the strongly kinked template strand. Four other crystal
structures with each of the canonical bases were also
solved in the editing mode, and the presence of three
nucleotides in the exonuclease site caused a shift in the
coordination state of its metal A from octahedral to
tetrahedral. Surprisingly, we find that all canonical bases
also enter the base-checking pocket with very small
differences in the binding geometry and in the calculated
binding free energy compared to deaminated ones. To explain
how this can lead to stalling of the replication fork, the
full catalytic pathway and its branches must be taken into
account, during which the base is checked several times.
Our results strongly suggest a switch from elongation to
editing modes right after nucleotide insertion when the
modified base is at position +5.
