Biology Reference
In-Depth Information
Cd
2
þ
—allosterically activates binding to DNA and, therefore, the metal works
as an obligate corepressor.
132,134
ScaR senses Mn
2
þ
like DtxR, but is, however,
structurally more similar to the Fe
2
þ
-dependent regulators DtxR and IdeR.
130
Two metal ions bind to
Mycobacterium tuberculosis
IdeR.
135
Binding to an
ancillary site promotes protein dimerization, but activation of DNA binding
requires both sites occupied by metal ions (
Fig. 7
C). The primary site is located
at the domain interface between the central dimerization and DNA-binding
domains of the protein, while the ancillary site is located between the dimer-
ization domain and a C-terminal domain that regulates the oligomerization
state of the protein and stabilizes the active conformation of the protein
(
Fig. 7
).
119,136-138
The primary metal-binding site, which is important for
DNA binding, is strictly conserved between ScaR and IdeR; however, this
site is empty in all the crystal structures of ScaR.
130
The ancillary site found
in IdeR is not conserved in ScaR, but a secondary metal-binding site about 5
˚
away is found in the structures of ScaR-Zn
2
þ
and ScaR-Cd
2
þ
. This secondary
site is novel in the DtxR/MntR family and is virtually identical to the Zn
2
þ
-
binding site identified in MutL, with the exception that the Zn
2
þ
metal ion is
fully coordinated by protein side chains (
Fig. 7
).
While the role and metal specificity of this novel metal-binding site found
in ScaR remain unclear, it has been postulated that His76 (or His79 in IdeR)
may play a role in coordinating the interactions between the two metal-binding
sites of the protein.
130
Therefore, it is plausible that His464 (the equivalent
residue in
B. subtilis
MutL) also plays a role in coordinating interactions
between the structural Zn
2
þ
and the catalytic metal of the protein. Accordingly,
the integrity of this histidine is critical for proficient DNA mismatch repair.
96,99
D. The Endonuclease Activity of MutL
Modrich and coworkers demonstrated that the endonuclease activity of
eukaryotic MutL
a
can be directed toward the discontinuous strand of a nicked
duplex in the presence of PCNA, RFC, and MutS
a
.
67,94
The primary role of
RFC is loading PCNA and hence it has only an indirect effect on the endonu-
clease activity of MutL
a
.
139
Conversely, PCNA and MutS
a
stimulate and
impose the mismatch dependency of the reaction, respectively, through their
direct interaction with MutL
a
.
139
In the absence of these protein factors,
MutL
a
retains a weak activity that nicks DNA in a nonspecific manner.
67
The
overall activity detected in this one-protein experiment is weak, but it has been
instrumental in characterizing the endonuclease activity of several prokaryotic
MutL proteins.
67,93-96
The endonuclease activity of MutL
a
is also stimulated by ATP, but not by
ADP, indicating that the conformational changes imposed by ATP binding are
important to activate the endonuclease activity of MutL (
Fig. 2
).
97,100,101
Sim-
ilar to eukaryotic MutL
a
, the DNA-nicking activity of
B. subtilis
MutL is
