Biology Reference
In-Depth Information
C terminus of the motif forms a short 3
10
helix that inserts the two aromatic
residues of the motif in hydrophobic pockets at the outer surface of the
clamp.
141-144
On the contrary, the
Q
IG
L
TD
F
motif found in hMLH1 is located
at the C terminus of helix
a
C and it is partially occluded from the solvent by
helix
a
A(
Fig. 5
). Given its conformation and the fact that the disruption of
this motif weakens, but does not abolish, the interaction with PCNA,
73
it is
likely that additional PCNA-interacting motifs are present in MutL
a
.
The direct interaction between human PMS2 (or yeast PMS1) with PCNA
has not been studied, but the specific interaction between the endonuclease
domain of
B. subtilis
MutL and
b
, the bacterial counterpart of PCNA, has been
demonstrated.
145
This interaction is mediated by the conserved
487
Q
EM
I
VP
motif, which resides in the external subdomain of the protein and is conspic-
uously exposed to the solvent (
Figs. 4 and 6
). The consensus sequence of this
motif resembles a
b
-binding motif
146
and its structure is virtually identical to
that seen in the structures of other
b
-binding peptides bound to
b
.
96
Mutations
within this motif abolish the interaction between the C-terminal domain of
B. subtilis
MutL and
b
and cause mismatch repair defects that are comparable
to those of null
mutL
strains.
96,145
As this motif is conserved in human PMS2
and other eukaryotic MutL proteins encompassing the endonuclase motif,
99
it
is presumed that it also mediates the interaction with PCNA.
The structure of the
b
sliding clamp from
B. subtilis
is unknown, but
superimposition of the endonuclease domain of
B. subtilis
MutL onto the
structure of the
b
clamp from another Gram-positive organism (
Streptococcus
pyogenes
) unveils two interesting facts. First, it suggests that the interaction of
one MutL protomer with the clamp would preclude the interaction of the
second protomer due to steric hindrance (
Fig. 8
). Consequently, only one
endonuclease site can be activated at a time even in homodimeric MutL
homologs, in turn preventing indiscriminate nicking. Second, the relative
orientation between
b
and MutL is such that the endonuclease site of MutL
aligns with the central chamber of the clamp, suggesting that
b
may stimulate
the endonuclease activity of MutL by either threading DNA to the active site or
tethering the endonuclease domain to DNA. The latter is the mechanism by
which PCNA stimulates the activity of the flap endonuclease Fen1.
147
The presence of PCNA also biases the nicking activity of human MutL
a
toward the discontinuous strand, an effect that is attributed to the directionality
of the loading.
139
PCNA and
b
are loaded at 3
0
double-strand-single-strand
junctions with a specific orientation.
142,148,149
Most proteins that bind to PCNA
or
b
, including MutL, do so through a hydrophobic cleft at the oligomerization
interface of the protein. The groove resides on one face of the clamp, often
referred to as the proximal face due to its proximity to the 3
0
end upon loading
and, therefore, the intrinsic asymmetry of the
b
-DNA complex is likely im-
posed onto the
b
-DNA-MutL complex.
96,139,145
