Covalently trapping MutS on DNA to study DNA mismatch recognition and signaling

Abstract

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...

Modern Aspects of the Structural and Functional Organization of the DNA Mismatch Repair System

“…In order to characterize these complexes, one can use a combination of various optical [95, 153, 154] and fluorescence [104] techniques associated with crosslinking of proteins to proteins and proteins to DNA [144]. A recently proposed approach to investigating short-lived complexes based on the covalent fixation of MutS to the DNA is considered to be rather promising [156]. …”