Figure 1.
The general scheme of MutS homodimer interaction with DNA.
The issues investigated in the current work are enclosed in the rectangles.
Figure 2.
45 bp duplexes V-X containing the variable nucleotide pair and the FRET pair.
Variable nucleotide pair is shown in bold. Alexa-594 (black circle) and Alexa-488 (grey circle) are linked to T residues.The duplexes are obtained by hybridization of three fragments (15-, 17- and 13-mer) on the 45-mer template strand. The nicks in the “bottom” strand of duplexes are indicated by vertical lines.
Table 1.
Characteristics of DNA duplex thermal stabilities.
Table 2.
Parameters of MutS binding to DNA, ATP hydrolysis and nucleotide exchange in ATPase domain of protein.
Table 3.
Change in energy transfer efficiency upon MutS binding to different DNA duplexes.
Figure 3.
The plasmid DNAs cleavage by MutH in a MutS-MutL dependent manner.
A, Analysis of the G/T-cccDNA treated with MutS-MutL-MutH mixture after 1, 5 or 10 min incubation in 1% agarose gel containing ethidium bromide. The initial cccDNA is shown (0 min). M – DNA ladder. B, Diagram representing the data of hydrolysis by MutS-MutL-MutH mixture of G/T-, G/C-, G/Tg- and A/Tg-cccDNA (the variable nucleotide pair introduced in cccDNA is indicated under the lanes) for 5 min. The experiments were performed 5 times. Error bars are standard deviations of the mean.
Figure 4.
Fluorescence emission spectra.
Panels A–F correspond to DNA duplexes V–X in the presence of MutS (400 nM per monomer – dashed line) or in the absence of protein (solid line). DNA duplexes (concentration 20 nM) contain FRET pair - Alexa-488 (donor) and Alexa-594 (acceptor). The central variable nucleotide pair in DNA is shown in parentheses. The samples were irradiated by light at 470 nm. Spectra were recorded at 500-800 nm. RU - the signal detector in stated units. Each spectrum was recorded at least three times. The figure shows one of the experiments.
Figure 5.
The MutS interaction with DNA duplexes containing FRET pair.
A, Models of MutS localization on DNA relative to fluorophores Alexa-594 (red) and Alexa-488 (green). The central nucleoside pair is indicated under the cartoons. MutS subunit A which interacts with the mismatch specifically is shown in blue-green; subunit B which forms only non-specific contacts with DNA is shown in yellow-red. B and C, Change in fluorescence anisotropy (Δr) upon maximal binding extent of MutS (total concentration per monomer 125 nM) to DNA (20 nM) containing various central nucleotide pairs: B, for Alexa-488; C, for Alexa-594. Error bars are standard deviations of the mean.