To avoid mutations in the genome, DNA replication is generally followed by DNA mismatch repair (MMR). MMR starts when a MutS homolog recognizes a mismatch and undergoes an ATP-dependent transformation to an elusive sliding clamp state. How this transient state promotes MutL homolog recruitment and activation of repair is unclear. Here we present a crystal structure of the MutS/MutL complex using a site-specifically crosslinked complex and examine how large conformational changes lead to activation of MutL. The structure captures MutS in the sliding clamp conformation, where tilting of the MutS subunits across each other pushes DNA into a new channel, and reorientation of the connector domain creates an interface for MutL with both MutS subunits. Our work explains how the sliding clamp promotes loading of MutL onto DNA, to activate downstream effectors. We thus elucidate a crucial mechanism that ensures that MMR is initiated only after detection of a DNA mismatch.DOI: http://dx.doi.org/10.7554/eLife.06744.001

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(Cytosine-5)-DNA methyltransferase SsoII (M.SsoII) functions as a methyltransferase and also as a transcription factor. Chemical and photochemical crosslinking was used for exploring the structure of M.SsoII-DNA complexes and M.SsoII in the absence of DNA. Photocrosslinking with 4-(N-maleimido)benzophenone demonstrated that in the M.SsoII complex with DNA containing the regulatory site, the M.SsoII region responsible for methylation was bound to DNA flanking the regulatory site, which contained no methylation sequence. This required high flexibility of the linker connecting the M.SsoII N-terminal domain and the M.SsoII region responsible for methylation. The flexibility was demonstrated by crosslinking with bis-maleimidoethane and 1,11-bis-maleimidotetraethyleneglycol.

The DNA mismatch repair (MMR) system recognizes and repairs errors that escaped the proofreading function of DNA polymerases. To study molecular details of the MMR mechanism, in vitro biochemical assays require specific DNA substrates carrying mismatches and strand discrimination signals. Current approaches used to generate MMR substrates are time-consuming and/or not very flexible with respect to sequence context. Here we report an approach to generate small circular DNA containing a mismatch (nanocircles). Our method is based on the nicking of PCR products resulting in single-stranded 3 overhangs, which form DNA circles after annealing and ligation. Depending on the DNA template, one can generate mismatched circles containing a single hemimethylated GATC site (for use with the bacterial system) and/or nicking sites to generate DNA circles nicked in the top or bottom strand (for assays with the bacterial or eukaryotic MMR system). The size of the circles varied (323 to 1100 bp), their sequence was determined by the template DNA, and purification of the circles was achieved by ExoI/ExoIII digestion and/or gel extraction. The quality of the nanocircles was assessed by scanning-force microscopy and their suitability for in vitro repair initiation was examined using recombinant Escherichia coli MMR proteins.

The DNA repair protein MutS forms clamp-like structures on DNA that search for and recognize base mismatches leading to ATP-transformed signaling clamps. In this study, the mobile MutS clamps were trapped on DNA in a functional state using single-cysteine variants of MutS and thiol-modified homoduplex or heteroduplex DNA. This approach allows stabilization of various transient MutS-DNA complexes and will enable their structural and functional analysis.The DNA mismatch repair system (MMR) detects and repairs errors that escaped the proofreading function of DNA polymerases. 1 The principal protein components of the bacterial MMR system are the homodimeric ATPases, MutS and MutL. In eukaryotes the MutS and MutL-homologues (MSH and MLH) are heterodimers, e.g. in humans MutSa (MSH2/MSH6) and MutLa (MLH1/PMS2).2 MMR is initiated when MutS recognizes a mismatch followed by ATP-induced complex formation with MutL.3 This ternary complex (DNAMutS-MutL) is a key active intermediate that couples mismatch recognition and discrimination of the template and nascent DNA strand. In E. coli the lack of adenine methylation in the nascent DNA strand at 5 0 -GATC-3 0 -sequences serves as a strand discrimination signal, 4 enabling the erroneous strand to be nicked by a third MMR protein, the monomeric endonuclease MutH. The nick is used by UvrD helicase and exonuclease, in the presence of single-strand DNA binding protein, to unwind and excise the erroneous strand until the mismatch is removed.DNA polymerase III and DNA ligase complete the repair process. In most bacteria and all eukaryotes that lack a MutH homologue, the strand discrimination signal is still unclear. However, it can be provided by pre-existing strand breaks or components of the replication machinery. 3During MMR, MutS forms several distinct complexes with DNA. First, MutS binds to DNA and searches for mismatches in a process involving linear diffusion. 5,6 Second, upon mismatch recognition MutS forms an asymmetric clamp-like complex in which the DNA is kinked by 45-601 at the mismatch region. 7-10DNA bending/kinking has been observed by atomic force microscopy 11 or Fo¨rster resonance energy transfer (FRET). 12,13Third, after mismatch recognition MutS undergoes ATP-induced conformational changes, finally leading to a long-lived complex with an ATP molecule bound to each subunit. 6,14,15 This 'sliding clamp' is believed to be the active form of bacterial MutS (or eukaryotic MutSa) that binds MutL and signals mismatch recognition to downstream events.Despite their functional importance, high-resolution structural data are not available for either the searching state or the signaling clamp state of MutS, in part due to little specific interaction between MutS and DNA in these complexes and their highly dynamic nature. To overcome these limitations we developed a covalent trapping strategy to capture MutS on DNA while searching (MutS bound to canonical DNA) or in the recognition state (MutS bound to mismatched DNA).Various methods have been established in the past to en...