Biology subjects

Laffeber, C.

Publications and source records attributed to Laffeber, C..

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

Experimental evidence for enhanced receptor binding by rapidly spreading SARS-CoV-2 variants

Rapidly spreading new variants of SARS-CoV-2 carry multiple mutations in the viral spike protein which attaches to the angiotensin converting enzyme 2 (ACE2) receptor on host cells. Among these mutations are amino acid changes N501Y (lineage B.1.1.7, first identified in the UK), and the combination N501Y, E484K, K417N (B.1.351, first identified in South Africa), all located at the interface on the receptor binding domain (RBD). We experimentally establish that RBD containing the N501Y mutation results in 9-fold stronger binding to the hACE2 receptor than wild type RBD. The E484K mutation does not significantly influence the affinity for the receptor, while K417N attenuates affinity. As a result, RBD from B.1.351 containing all three mutations binds 3-fold stronger to hACE2 than wild type RBD but 3-fold weaker than N501Y. The recently emerging double mutant E484K/N501Y binds as tight as N501Y. The independent evolution of lineages containing mutations with different effects on receptor binding affinity, viral transmission and immune evasion underscores the importance of global viral genome surveillance and functional characterization.

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↗