Biology subjects

Friedhoff, P.

Publications and source records attributed to Friedhoff, P..

3 recordsLinked to original sources

bioRxiv2021

DNA mismatch and damage detection using a FRET-based assay for monitoring the loading of multiple MutS sliding clamps

We developed a sensitive, homogeneous fluorescence assay for the detection of DNA mismatches and DNA damage based on the mismatch repair (MMR) protein MutS. The assay is based on Forster resonance energy transfer (FRET) between SYBR Green I (SG), non-covalently bound to DNA, and Alexa Fluor 647 (AF647) conjugated to MutS. In contrast to previous assays using only the mismatch binding activity of MutS, we exploited the ATP-dependent loading of multiple MutS sliding clamps provoked by mismatch/damage to the DNA, which increases the overall sensitivity of the assay. The assay was validated using a well-characterized 3 kb circular DNA containing a single G/T mismatch. We also demonstrate that treatment of long (multiple kb) DNA with various chemical or physical agents including non-denaturing bisulfite conversion of cytosine to uracil, cisplatin modification or ultraviolet light (UVC) results in changes in the DNA that can be detected by the FRET-based MutS biosensor.

biochemistry↗

bioRxiv2021

Cryo-EM structures reveal how ATP and DNA binding in MutS coordinate the sequential steps of DNA mismatch repair

DNA mismatch repair detects and removes mismatches from DNA reducing the error rate of DNA replication a 100-1000 fold. The MutS protein is one of the key players that scans for mismatches and coordinates the repair cascade. During this, MutS undergoes multiple conformational changes that initiate the subsequent steps, in response to ATP binding, hydrolysis, and release. How ATP induces the different conformations in MutS is not well understood. Here we present four cryo-EM structures of Escherichia coli MutS at sequential stages of the ATP hydrolysis cycle. These structures reveal how ATP binding and hydrolysis induces a closing and opening of the MutS dimer, respectively. Additional biophysical analysis furthermore explains how DNA binding modulates the ATPase cycle by preventing hydrolysis during scanning and mismatch binding, while preventing ADP release in the sliding clamp state. Nucleotide release is achieved when MutS encounters single stranded DNA that is produced during the removal of the daughter strand. This way, the combination of the ATP binding and hydrolysis and its modulation by DNA enable MutS to adopt different conformations needed to coordinate the sequential steps of the mismatch repair cascade.

biochemistry↗

bioRxiv2018

The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair

DNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone.

biochemistry↗