Conformational changes both in DNA and MutS during the initial steps of DNA mismatch repair system and during the ATPase cycle were analyzed using state-of-the-art fluorescence techniques, down to the single molecule level.
MutS binds mismatches with preferred orientations:
MutS scans the DNA for base-base mismatches and small insertion-deletion loops. A hallmark of the mismatch recognition mechanism is DNA kinking by 45º-60º as observed in the co-crystal structure of MutS-DNA complexes from bacteria to man. The change in the distance of two positions in the DNA upon kinking was exploited by FRET (Fluorescence Resonance Energy Transfer). 16 different 42bp oligonucleotides containing all possible base-base in a central position, with an acceptor fluorophore on the top strand and a donor on the bottom strand, were tested for DNA binding and kinking by MutS using an in-solution FRET assay. Single molecule techniques were used to analyze the binding and bending modes of MutS at highest resolution. The results demonstrate that MutS binds certain mismatches with a preferred orientation. The preferential binding orientation may have important impact on the coupling of mismatch repair and replication, in particular on the mechanism involving directed loading of the heterodimeric MutSalpha by interaction with the replication factor PCNA.
Nucleotide influence of the DNA binding:
The double labeled G:T oligonucleotide was used to determine the binding and bending kinetics of MutS (member of the ABC ATPase family) to DNA in the presence of ADP, ATP and ADPnP. In addition, the influence of nucleotide on the MutS-induced DNA bending was analyzed using single-molecule techniques to determine the bent/kinked populations present with each nucleotide. The results showed that the association of MutS to DNA in the presence of ADP involves at least a two step mechanism, possibly a fast binding/bending step followed by a kinking at the mismatch. A quantitative analysis of the FRET population showed that the DNA with MutSADP almost homogeneous forming mainly a kinked/bent complex. In contrast, complex formed in the absence of nucleotide or in the presence of ATP are more heterogenous involving at least two complexes, one of which is kinked/bent whereas the others are either unbent or bound not at the mismatch. Pre-steady state kinetic analysis of MutS-DNA association in the presence of ATP and MutS-DNA dissociation in the presence of ATP, ADP or ADPnP revealed distinct phases depending on the nucleotide state of the starting complex. Communication between ATPase domain and clamp domain:
In the present work conformational changes in MutS were monitored using a FRET analysis employing single-cysteine variants of MutS. To simplify the data analysis a fully functional single-cysteine dimer variant of MutS was generated thereby avoiding complication due to the formation of tetramers in case of wild type MutS. The work presented here shows that single-cysteine mutants of MutS could be fluorescently labeled with one or two fluorophores suitable for FRET analysis without affecting the function of the protein, e.g. in DNA binding, mismatch recognition and mismatch-provoked MutH activation. Sedimentation velocity analysis and in-solution FRET measurements demonstrated that the fluorescently labeled MutS forms stable dimers in the presence of DNA or nucleotide. Binding of the non-hydrolyzable ATP-analogue ADPnP resulted in a closed, compact form of MutS which is unable to bind to DNA. The dynamics of conformational changes in the clamp domain upon DNA and/or nucleotide binding were monitored using stopped-flow. Based on these data the clamp domain of MutS exists in at least four different states. The data obtained from the fluorescence analysis using double labeled DNA, labeled DNA/labeled MutS and double labeled MutS were included in model for the DNA binding and ATPase cycle of MutS.
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