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. 2014 Aug 18;9(8):e104963.
doi: 10.1371/journal.pone.0104963. eCollection 2014.

Is thymidine glycol containing DNA a substrate of E. coli DNA mismatch repair system?

Affiliations

Is thymidine glycol containing DNA a substrate of E. coli DNA mismatch repair system?

Svetlana A Perevozchikova et al. PLoS One. .

Erratum in

Abstract

The DNA mismatch repair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for E. coli MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the E. coli MMR system. Probably, the MutS transformation to active "sliding clamp" conformation on Tg-DNA is problematic.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
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
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.
Figure 3
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
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
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.

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