Biology Reference
In-Depth Information
connecting strands
8. Phe111 is wedged between the base opposite the
lesion (a cytosine) and the neighboring base, and causes unstacking of these
bases, leading to the severe kinking of the DNA. Additionally, Arg109 forms
H-bonds with the opposite base, leading to discrimination against A as the
opposite base.
55,74
The analogous residues in MvNei1 (Leu84, Arg114, and Phe116),
61
human
NEIL1 (Met81, Arg118, and Phe120),
67
and
Arabidopsis thaliana
Fpg
(AthFpg) (Met78, Arg126, and Phe128) (St
´
phanie Duclos, Pierre Aller,
Pawel Jaruga, Miral Didzaroglu, Susan S. Wallace and Sylvie Doubli
´
, Manu-
script submitted to DNA Repair) are similar to those seen in the bacterial Fpg
proteins, which discriminate against A as a base opposite the lesion.
74
In
contrast, EcoNei inserts three consecutive residues Gln69, Leu70, and Tyr71
into the void created upon base extrusion.
56
The three residues are located on a
loop connecting
b
7and
b
5. Tyr71 is wedged between the orphaned base and its
3
0
neighbor and stabilizes the severely kinked DNA. Two of the void-filling
residues are lacking in MmuNeil3, which appears to be related to the prefer-
ence of this enzyme for single-stranded DNA (Minmin Liu, Kayo Imamura,
Sylbie Doublie and Susan S. Wallace, manuscript in preparation).
In addition to the void-filling residues, the Fpg/Nei proteins contain an
absolutely conserved Lys residue (Lys57 in EcoFpg) and a conserved Asn
(Asn169 in EcoFpg), which is part of the H2THmotif.
55
Lys57 forms salt-bridges
with P
-1
and P
-2
while Asn169 forms bonds through backbone and side-chain
amides to P
-1
and P
0
of the DNA (
Fig. 2
B and C). Another highly conserved
residue in the Fpg/Nei family of proteins is Arg259 (in EcoFpg), which is part of
the zinc finger motif and is involved in the formation of salt bridges with the
phosphodiester backbone
55
(
Fig. 2
B). In the bacterial Fpg and eukaryotic Fpg/
Nei proteins, there is no gross conformational change in the overall domain
structure upon DNA binding.
55,57,59,64,72
On the other hand, the side-chains of
the void-filling residues and conserved residues in the hallmark motifs show
small changes. The development of disulfide-crosslinking techniques used to
study the structure of BstFpg bound to lesion-containing DNA and undamaged
DNA indicates that the enzyme possesses intrahelical recognition of the damage
and can detect the subtle differences between the damaged base and its unda-
maged counterpart even at an initial encounter (Refs.
64,65
and see below).
Comparing 8-oxoG with guanine in DNA suggests that the enzyme induces a
local conformational change in the DNA backbone in which the sugar pucker
(C2
0
-endo) adopts a different conformation (C4
0
-exo) to prevent a steric clash
between the 8-oxo group of 8-oxoG and the C2
0
of the sugar.
64
A loop region (called the
b
4 and
b
-helical C-terminal domain
of the Fpg proteins has been presumed to be involved in lesion recognition. In
the unliganded structure of TthFpg, this lesion recognition loop is ordered,
54
but in structures of BstFpg bound to DNA containing an AP site, the density
for this loop disappears, suggesting that this region is disordered.
57
a
F-
b
9/10 loop) in the
a
In the
