Biology Reference
In-Depth Information
(
Figs. 4 and 5
). This is not surprising considering that the C-terminal region of
hMLH1 (residues 475-756) cannot be aligned with any other MutL para-
log.
42,96,98,99
As for other MutL homologs, the dimerization interface of
hMLH1 is still defined by a four-stranded antiparallel
b
-sheet. However, the
domain adopts a compact and predominantly helical structure that cannot
be easily divided into subdomains. This compact structure is primarily caused
by the N terminus of the domain wrapping around helix
a
C—the equivalent of
helix
a
A on the structures of
B. subtilis
and
E. coli
MutL, the insertion of helix
a
E, and the extension of helix
a
F(
Fig. 5
). Additionally, the relative orientation of
helices
a
C,
a
E, and
a
F(
a
A,
a
C, and
a
D, respectively, in other MutL-CTD
structures) is markedly different. It is conceivable that these differences are
important to form functional MutL
a
,MutL
b
, and MutL
g
heterodimers. Indeed,
trypsin protection assays suggest that formation of a stable hMutL
a
heterodimer
requires a longer fragment of PMS2 than the region predicted from sequence
and structural homology (J. W. and A. G., unpublished results).
96
III. Architecture of the Endonuclease Domain
In addition to the endonuclease motif, five highly conserved motifs have
been identified within the C-terminal domain of MutL.
99
Four of them (GQ,
[A/S]C[K/R], C[P/N]HGRP, and FXR) are found exclusively in MutL homo-
logs that contain the conserved endonuclease motif (DQHAX
2
EX
4
E), whereas
the fifth (QXXL[L/I]XP) is found in MutL homologs that have, and also in those
that do not have, endonuclease activity.
99
It was predicted that the four
additional motifs identified in MutL proteins carrying the conserved endonu-
clease motif would define a single active site,,
99,115
and the structures of the C-
terminal domains of
B. subtilis
and
N. gonorrhoeae
MutL have recently con-
firmed this prediction.
96,114
A. The Endonuclease Site
The endonuclease site of MutL is a composite active site where the
DQHAX
2
EX
4
E, [A/S]C[K/R], and C[P/N]HGRP motifs are provided by one
MutL protomer, while the FXR motif is contributed by the other protomer of
the dimer (
Fig. 6
).
96
Accordingly, this motif is absent in eukaryotic MutL
homologs bearing endonuclease activity, but present in yeast and human
MLH1.
99
Except for the [A/S]C[K/R] motif, contributed by the external sub-
domain, all the motifs that define the endonuclease site reside in the dimer-
ization subdomain of the protein (
Fig. 6
).
Aquifex aeolicus
MutL, which lacks the external subdomain, is a proficient
endonuclease, suggesting that this subdomain of the protein has minimal effect
on the endonuclease activity of MutL (
Fig. 4
).
93,116
A. aeolicus
MutL has the
