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.

Thermodynamic evaluation of the impact of DNA mismatches in PCR-type SARS-CoV-2 primers and probes

BackgroundDNA mismatches can affect the efficiency of PCR techniques if the intended target has mismatches in primers or probes regions. The accepted rule is that mismatches are detrimental as they reduce the hybridization temperatures, yet a more quantitative assessment is rarely performed.MethodsWe calculate the hybridization temperatures of primer/probe sets after aligning to SARS-COV-2, SARS-COV-1 and non-SARS genomes, considering all possible combinations of single, double and triple consecutive mismatches. We consider the mismatched hybridization temperature within a range of 5 °C to the fully matched reference temperature.ResultsWe obtained the alignments of 19 PCR primers sets that were recently reported for the detection of SARS-CoV-2 and to 21665 SARS-CoV-2 genomes as well as 323 genomes of other viruses of the coronavirus family of which 10 are SARS-CoV-1. We find that many incompletely aligned primers become fully aligned to most of the SARS-CoV-2 when mismatches are considered. However, we also found that many cross-align to SARS-CoV-1 and non-SARS genomes.ConclusionsSome primer/probe sets only align substantially to most SARS-CoV-2 genomes if mismatches are taken into account. Unfortunately, by the same mechanism, almost 75% of these sets also align to some SARS-CoV-1 and non-SARS viruses. It is therefore recommended to consider mismatch hybridization for the design of primers whenever possible, especially to avoid undesired cross-reactivity.

Overexpression of MutS impairs DNA mismatch repair and causes cell division defect in E.coli

MutS and its homologues, from prokaryotes to humans, recognize and bind to DNA mismatches generated during DNA replication, initiate DNA mismatch repair and ensures 100-200 fold increase in replication fidelity. In E.coli, through post transcriptional regulation, at least three mechanisms mediate decline of MutS intracellular concentrations during stress conditions. To understand the significance of this multifold regulation, we overexpressed MutS in E.coli and found that it led to impairment of DNA mismatch repair as reflected by preferential accumulation of transition mutations in spontaneous base pair substitution spectrum. This phenomenon was dependent on MutS-mismatch affinity and interaction. Higher MutS overexpression levels promoted DNA double strand breaks, inhibited cell division and resultantly caused a manifold increase in E.coli cell length. This cell division defect involved a novel MutS-FtsZ interaction and impediment of FtsZ ring function. Our findings may have relevance for cancers where mismatch proteins are known to be overexpressed.

DNA ligase I variants fail in the ligation of mutagenic repair intermediates with mismatches and oxidative DNA damage

DNA ligase I (LIG1) joins DNA strand breaks during DNA replication and repair transactions and contributes to genome integrity. The mutations (P529L, E566K, R641L and R771W) in LIG1 gene are described in patients with LIG1-deficiency syndrome that exhibit immunodeficiency. LIG1 senses 3’-DNA ends with a mismatch or oxidative DNA base inserted by a repair DNA polymerase. However, the ligation efficiency of the LIG1 variants for DNA polymerase-promoted mutagenesis products with 3’-DNA mismatches or 8-oxo-2’-deoxyguanosine (8-oxodG) remains undefined. Here, we report that R641L and R771W fail in the ligation of nicked DNA with 3’-8-oxodG, leading to an accumulation of 5’-AMP-DNA intermediates in vitro. Moreover, we found that the presence of all possible 12 non-canonical base pairs variously impacts the ligation efficiency by P529L and R771W depending on the architecture at the DNA end, whereas E566K exhibits no activity against all substrates tested. Our results contribute to the understanding of the substrate specificity and mismatch discrimination of LIG1 for mutagenic repair intermediates and the effect of non-synonymous mutations on ligase fidelity.