4 research outputs found
Large conformational changes in MutS during DNA scanning, mismatch recognition and repair signalling: Conformations of MutS during DNA MMR activation
MutS protein recognizes mispaired bases in DNA and targets them for mismatch repair. Little is known about the transient conformations of MutS as it signals initiation of repair. We have used single-molecule fluorescence resonance energy transfer (FRET) measurements to report the conformational dynamics of MutS during this process. We find that the DNA-binding domains of MutS dynamically interconvert among multiple conformations when the protein is free and while it scans homoduplex DNA. Mismatch recognition restricts MutS conformation to a single state. Steady-state measurements in the presence of nucleotides suggest that both ATP and ADP must be bound to MutS during its conversion to a sliding clamp form that signals repair. The transition from mismatch recognition to the sliding clamp occurs via two sequential conformational changes. These intermediate conformations of the MutS:DNA complex persist for seconds, providing ample opportunity for interaction with downstream proteins required for repair
Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes
Dynamics of MutSâMismatched DNA Complexes Are Predictive of Their Repair Phenotypes
MutS
recognizes baseâbase mismatches and base insertions/deletions
(IDLs) in newly replicated DNA. Specific interactions between MutS
and these errors trigger a cascade of proteinâprotein interactions
that ultimately lead to their repair. The inability to explain why
different DNA errors are repaired with widely varying efficiencies <i>in vivo</i> remains an outstanding example of our limited knowledge
of this process. Here, we present single-molecule FoÌrster resonance
energy transfer measurements of the DNA bending dynamics induced by <i>Thermus aquaticus</i> MutS and the E41A mutant of MutS, which
is known to have error specific deficiencies in signaling repair.
We compared three DNA mismatches/IDLs (T-bulge, GT, and CC) with repair
efficiencies ranging from high to low. We identify three dominant
DNA bending states [slightly bent/unbent (<b>U</b>), intermediately
bent (<b>I</b>), and significantly bent (<b>B</b>)] and
find that the kinetics of interconverting among states varies widely
for different complexes. The increased stability of MutSâmismatch/IDL
complexes is associated with stabilization of <b>U</b> and lowering
of the <b>B</b> to <b>U</b> transition barrier. Destabilization
of <b>U</b> is always accompanied by a destabilization of <b>B</b>, supporting the suggestion that <b>B</b> is a ârequiredâ
precursor to <b>U</b>. Comparison of MutS and MutS-E41A dynamics
on GT and the T-bulge suggests that hydrogen bonding to MutS facilitates
the changes in baseâbase hydrogen bonding that are required
to achieve the <b>U</b> state, which has been implicated in
repair signaling. Taken together with repair propensities, our data
suggest that the bending kinetics of MutSâmismatched DNA complexes
may control the entry into functional pathways for downstream signaling
of repair