Biology Reference
In-Depth Information
responsible for the transfer of an early Fpg/Nei gene to early eukaryotes in
which these Fpg/Nei homologs led to the diversification of the Fpg/Nei pro-
teins in higher eukaryotes.
34,52,53
III. Fpg/Nei Structures
A. Introduction
Over the past decade, there has been a significant increase in the number
of crystal structures of Fpg/Nei glycosylases.
54-69
The advent of techniques
such as reductive crosslinking using sodium borohydride has played an essen-
tial role in trapping stable protein-DNA complexes for the purposes of crys-
tallization and for the elucidation of the mechanism and role of these intricate
enzymes (Refs.
54-56,64-66,70,71
; for reviews see Refs.
70,71
). Other ap-
proaches successfully used to produce stable glycosylase-DNA complexes
include the generation of site-directed mutants of active site residues to abolish
catalysis and the use of noncleavable substrates such as tetrahydrofuran (THF),
which mimics an abasic (AP) site,
60,72
and noncleavable cyclopentane FapyG
(cFapyG).
68
A summary of all the currently available crystal structures of the
Fpg/Nei family of glycosylases and their substrate preferences is listed in
Table I
. Crystal structures of Fpg proteins from various bacterial species such
as
Thermus thermophilus
(Tth) Fpg (without DNA),
54
Escherichia coli
(EcoFpg),
55
Geobacillus stearothermophilus
Fpg (BstFpg),
57,59,64-66
and
Lactococcus lactis
Fpg (LlaFpg)
58,60,63,68,69
complexed with DNA substrates
have been determined. The Fpg-DNA complexes include Schiff base inter-
mediates, noncovalent complexes with AP site analogs, and recognition or end
product complexes. Although the structure of EcoNei as a Schiff base inter-
mediate in a complex with DNA was solved,
56
it wasn't until recently that the
unliganded structure of EcoNei was determined which revealed a unique
interdomain conformational change upon DNA binding.
62
Furthermore, the
structures of unliganded human NEIL1,
61
unliganded Mimivirus Neil1
(MvNei1), and MvNei1 in a complex with THF were subsequently obtained.
67
The first crystal structures of an Nei bound to damaged bases were recently
reported: MvNei1 was captured in a complex with DNA containing either
thymine glycol (Tg) or 5-hydroxyuracil (5-OHU).
104
Overall, the structures of the Fpg and Nei proteins are similar, with a distinct
2-domain architecture connected by a flexible hinge region (
Fig. 1
AandBusing
EcoFpg and EcoNei as examples).
55,56,72
In general, the N-terminal region is
predominantly
b
-sheet-rich and is composed of a
b
-sandwich flanked by
a
-helices, two of which form a
conserved H2TH motif, as well as two antiparallel
-helices. The C-terminal domain comprises
a
-strands that fold into a zinc
finger motif. These signature motifs are characteristic of both Fpg and Nei
b
