Nucleic Acids Research

MutS initiates mismatch repair by recognizing mismatches in newly replicated DNA. Specific interactions between MutS and mismatches within double-stranded DNA promote ADP-ATP exchange and a conformational change into a sliding clamp. Here, we demonstrated that MutS from Pseudomonas aeruginosa associates with primed DNA replication substrates. The predicted structure of this MutS-DNA complex revealed a new DNA binding site, in which Asn 279 and Arg 272 appeared to directly interact with the 3-OH terminus of primed DNA. Mutation of these residues resulted in a noticeable defect in the interaction of MutS with replication DNA substrates. Remarkably, MutS interaction with a mismatch within primed DNA induced a compaction of the protein structure and impaired the formation of an ATP-bound sliding clamp. Our findings reveal a novel DNA binding mode, conformational change and intramolecular signaling for MutS recognition of mismatches within DNA replication structures.

As an alternative to historical enzyme isolation, metagenomic databases (e.g. MGnify) provide information on vast unculturable microbial diversity, especially from extreme environments, and constitute an enormous source of functional proteins. Conservative mining of these data by close sequence homology alone tends to identify merely different versions of known enzymes. Here we present a discovery strategy of meganucleases based on wider capture of less homologous enzymes with new function in metagenomic databases, incorporating metadata with homology, relying on cell-free expression to bypass host incompatibility and the need for purification, along with using deep sequencing for experimental assessment of substrate specificity and cleavage pattern, circumventing classical gel-based profiling. Specifically, we discovered the temperature-stable (>55{degrees}C), intron-encoded LAGLIDADG meganuclease I-MG11 that recognizes a 17 base pair sequence to generate unique 4 base pair palindromic 3'-overhangs -- the first monomeric meganuclease to produce such overhangs. Co-folding models of I-MG11 bound to DNA provide a structural context for enzyme-DNA interactions, highlighting differences from other monomeric LAGLIDADG meganucleases (e.g. I-SceI) shaped by InDels (insertion-deletions) in the DNA binding region that may cause specificity changes. Our strategy streamlines bona fide identification and annotation of meganucleases, while the unique properties of I-MG11 expand the molecular biology toolbox. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=31 SRC="FIGDIR/small/712669v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@885de7org.highwire.dtl.DTLVardef@cce9fdorg.highwire.dtl.DTLVardef@116055borg.highwire.dtl.DTLVardef@b9b59e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Transfer RNA (tRNA) molecules serve as essential adapters during protein translation. While direct RNA sequencing (DRS) via Oxford Nanopore Technologies has emerged as a powerful platform for systematic tRNAome profiling, we currently lack a simple and robust statistical framework for nanopore tRNA data analyses. Here, we address this gap by developing AMaNITA (Abundance, Modifications, and Nanopore Intensity Toolbox Application), an end-to-end bioinformatic workflow that enables simplified, robust, and scalable analyses of nanopore native tRNA sequencing datasets. AMaNITA streamlines the entire analytical trajectory: from upstream processing (basecalling, mapping, filtering, batch effect correction) to downstream assessment of differential tRNA abundance and modification stoichiometry. The workflow generates an interactive HTML report for data exploration and analysis, allowing the user to download the source data files and resulting plots. AMaNITA can be executed using Singularity from the command line, without requiring installation of dependencies.

RNA molecules undergo a number of chemical modifications whose effects can alter their structure and molecular interactions. Previous studies have shown that RNA editing can impact the formation of ribonucleoprotein complexes and influence the assembly of membrane-less organelles such as stress-granules. For instance, N6-methyladenosine (m6A) enhances SG formation and N1-methyladenosine (m1A) prevents their transition to solid-like aggregates. Yet, very little is known about adenosine to inosine (A-to-I) modification that is very abundant in human cells and not only impacts mRNAs but also non-coding RNAs. Here, we built the CROSSalive predictor of A-to-I effects on RNA structure based on high-throughput in-cell experiments. Our method shows an accuracy of 90% in predicting the single and double-stranded content of transcripts and identifies a general enrichment of double-stranded regions caused by A-to-I in long intergenic non-coding RNAs (lincRNAs). For the individual cases of NEAT1, NORAD and XIST, we investigated the relationship between A-to-I editing and interactions with RNA-binding proteins using available CLIP data. We found that A-to-I editing is linked to alteration of interaction sites with proteins involved in phase-separation, which suggests that RNP assembly can be influenced by A-to-I. CROSSalive is available at http://service.tartaglialab.com/new_submission/crossalive.

Deinococcus radiodurans harbors a largely classical bacterial DNA repair machinery yet displays exceptional resistance to ultra-violet and ionizing radiation. To investigate whether crosstalk between its DNA repair pathways contributes to this phenotype, we mapped putative interactions between the nucleotide excision repair (NER) and base excision repair (BER) pathways, which together are responsible for the removal of nucleobase lesions. Using a bacterial two-hybrid system, we identified multiple direct interactions between NER and BER proteins, notably involving the two UvrA variants, and validated these interactions in vitro. Furthermore, functional analyses revealed that NER interferes with the BER-mediated removal of oxidized guanines by the Fpg DNA glycosylase, likely through competition for DNA binding and sequestration of Fpg. Finally, UvrB and UvrC were found to further process the Fpg incision product in an ATP-dependent, UvrA1-independent manner. Together, these results demonstrate a multi-level crosstalk between NER and BER in D. radiodurans, which may contribute to its extraordinary DNA repair capacity. To our knowledge, this represents the first evidence of such a complex interplay in bacteria.

Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is a key enzyme in the Base Excision Repair pathway, responsible for processing abasic (AP-) sites. Recent studies revealed that APE1 participates in repairing DNA secondary structures as G-quadruplexes (G4). Telomeres, stabilized by shelterin proteins, are rich in G4, where APE1 binds and repairs AP-sites to maintain their integrity. The complementary cytosine-rich strand forms another structure, the i-motif (iM), essential for telomere maintenance, though its repair mechanism remains unclear. Herein we investigate APE1 binding and processing capabilities toward native and damaged telomeric iM, bearing AP-sites in different positions. Using biochemical and biophysical assays, we found that APE1 binds the telomeric iM-sequence and that its cleavage efficiency depends on AP-site position within iM. Proximity Ligation Assay analysis, in HeLa and U2OS cells, highlighted a novel interaction between APE1 and PCBP1, a well-known iM-folding modulator. PCBP1 binds iM with higher affinity than APE1 and inhibits its cleavage activity on damaged iM. Immunofluorescence and Telomere Restriction Fragment analyses showed that depletion of APE1 or PCBP1 impairs their interaction with the shelterin components, affecting telomere length. These results connect APE1 canonical DNA repair activity with the maintenance of non-canonical DNA secondary structures in telomeres, through its interaction with PCBP1. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/694817v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1962c63org.highwire.dtl.DTLVardef@3c3f60org.highwire.dtl.DTLVardef@164d78borg.highwire.dtl.DTLVardef@1831aae_HPS_FORMAT_FIGEXP M_FIG C_FIG

For many years, the genetic manipulation of mitochondrial DNA was largely hampered by inefficient delivery of nucleic acids to mitochondria. However, the development of mitoCBEs, such as mitochondrial cytosine base editors (DdCBEs), which catalyse C*G-to-T*A conversions, and more recently, mitoABEs, such as transcription-activator-like effector (TALE)-linked deaminases (TALEDs) enabling A*T-to-G*C conversion, has transformed this field. Generally, mitochondrial base editors exhibit high on-target efficiency and are straightforward to design and use. Nonetheless, unintended off-target effects cannot be overlooked and should be assessed consistently with each experiment, which can be challenging without specialised bioinformatic expertise. Here, we introduce Mitochondrial Base Editor Analysis Package (MitoBEAP), which, to our knowledge, is the first R package specifically designed to analyse next-generation sequencing data from base-edited mtDNA samples. The package facilitates the analysis of potential off-target effects, offers multiple visualisation options, and allows customisation of graphics and thresholds for calculations. As a proof of concept, this study demonstrates how MitoBEAP can be utilised to measure the efficiency of DdCBE treatment targeting human 12S rRNA, as well as to identify potentially harmful off-target conversions across the mtDNA.

The human DNA repair factor CtIP helps to initiate the resection of double-stranded DNA breaks for repair by homologous recombination, in part through its ability to bind and bridge DNA molecules. However, CtIP is a natively disordered protein that bears no apparent similarity to other DNA-binding proteins and so the structural basis for these activities remains unclear. In this work, we have used bulk DNA binding, single molecule tracking, and DNA bridging assays to study wild-type and variant CtIP proteins to better define the DNA binding domains and the effects of mutations associated with inherited human disease. Our work identifies a monomeric DNA-binding domain in the C-terminal region of CtIP. CtIP binds non-specifically to DNA and can diffuse over thousands of nucleotides. CtIP-mediated bridging of distant DNA segments is observed in single-molecule magnetic tweezers experiments. However, we show that binding alone is insufficient for DNA bridging, which also requires tetramerization via the N-terminal domain. Variant CtIP proteins associated with Seckel and Jawad syndromes display impaired DNA binding and bridging activities. The significance of these findings in the context of facilitating DNA break repair is discussed. Significance StatementCtIP helps to repair broken chromosomes through its ability to bind and bridge DNA molecules. We studied the structural and biochemical basis for these activities and how they are affected by hereditary CtIP mutations associated with developmental disorders. We discovered a minimal domain in the C-terminal region of CtIP which supports DNA binding as a monomer. DNA binding is non-specific and facilitates 1D diffusion, but binding alone is insufficient for intermolecular tethering of DNA molecules which requires tetramerization of CtIP via N-terminal coiled-coil domains. All disease variants tested displayed impaired DNA bridging activity. These results have important implications for understanding the role of CtIP as a hub protein for DNA break repair and its dysfunction in human disease.

DNA topology is a key regulator of chromatin structure and transcription, yet its direct role in transcription factor recognition remains unclear. Here, we investigate how distinct DNA topological states modulate binding of the Saccharomyces cerevisiae bZIP transcription factor GCN4 using topologically defined plasmids. By combining, complementary biochemical approaches, including Bio-Layer Interferometry applied here for the first time to topology-dependent protein-DNA interactions, we show that DNA supercoiling directly reshapes GCN4-DNA recognition. Positively supercoiled DNA forms more stable and persistent complexes, whereas negatively supercoiled DNA retains greater conformational heterogeneity. To interpret these effects, we performed multiscale molecular simulations. Coarse-grained simulations of plasmids recapitulate the global topology-dependent trends observed experimentally, while matched minicircle models reproduce the same behaviour at the local scale. In strong agreement with experimental data, simulations reveal that DNA topology modulates the conformational ensemble of the GCN4 basic region. Overall, positively supercoiled DNA promotes a more ordered binding mode and localized protein distribution, whereas negatively supercoiled DNA supports increased structural plasticity. These findings identify DNA topology as an active determinant of transcription factor recognition and provide a multiscale framework linking global DNA mechanics to local protein-DNA interactions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/722604v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@18f8ba9org.highwire.dtl.DTLVardef@11a395dorg.highwire.dtl.DTLVardef@ac093borg.highwire.dtl.DTLVardef@923212_HPS_FORMAT_FIGEXP M_FIG C_FIG

DNA-protein interactions are essential in all aspects of every living cell. Understanding of how features embedded in the DNA sequence affect specific interactions with proteins is challenging but important, since it may contribute to finding the means to regulate metabolic pathways involving DNA-protein interactions. Using a massive experimental benchmark dataset of binding scores for DNA sequences and a machine learning workflow, we describe the binding to DNA of T7 primase, as a model system for specific DNA-protein interactions. Effective binding of T7 primase to its specific DNA recognition sequences triggers the formation of RNA primers that serve as Okazaki fragment start sites during DNA replication.

Pentatricopeptide repeat (PPR) proteins are eukaryotic RNA binding proteins with multiple roles in mitochondrial and chloroplast transcript processing. PPR proteins are naturally modular and hold great potential for development into tools for RNA processing or controlling RNA folding or expression. However, construction of synthetic PPR proteins is challenging due to their highly repetitive sequences. Here, we present the GRASP kit for assembly of synthetic PPR proteins. Utilising the S-variant of PPR motifs, we designed a library of 42 plasmids which can be combined to assemble synthetic PPR proteins with 9, 14 or 19 motifs to target any RNA sequence of the same length. The GRASP kit enables rapid design and construction of PPR proteins of any desired specificity and is compatible with the MoClo assembly standard. To demonstrate the capabilities of GRASP, we assembled a synthetic PPR RNA editing protein and variants with altered sequence specificity. We tested the functionality of 31 synthetic PPR protein variants against a set of 46 RNA targets and used RNA sequencing to determine levels of RNA editing. The variations in editing provide a wealth of insights into PPR-RNA interactions. The GRASP kit provides a foundation for further development of synthetic PPR protein technologies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/661641v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@d8c1d2org.highwire.dtl.DTLVardef@9ccba8org.highwire.dtl.DTLVardef@1feadfeorg.highwire.dtl.DTLVardef@150ffb7_HPS_FORMAT_FIGEXP M_FIG C_FIG

In the last decade, several novel functions of the mammalian Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APE1) have been discovered, going far beyond its canonical function as a DNA repair enzyme, unveiling its potential roles in cancer development. Indeed, it was shown to be involved in DNA G-quadruplex biology and RNA metabolism, most importantly in the miRNA maturation pathway and the decay of oxidized- or abasic-miRNAs during oxidative stress conditions. Furthermore, in recent years several non-canonical pathways of miRNA biogenesis have been described, with a specific focus on guanosine-rich precursors that can form RNA G-quadruplex (rG4) structures. In this study, we show that several miRNA precursors, dysregulated upon APE1-depletion, contain an rG4 motif and that their corresponding target genes are upregulated after APE1-depletion. We also show, both by in vitro assays and by using a HeLa cell model, that APE1 can bind and regulate the folding of an rG4 structure contained in pre-miR92b, with a mechanism strictly dependent on critical lysine residues present in the N-terminal disordered region. Furthermore, APE1 depletion in HeLa cells alters the maturation process of miR-92b, mainly affecting the shuttling between the nucleus and cytosol. Lastly, bioinformatic analysis of APE1-regulated rG4-containing miRNAs supports the relevance of our findings for cancer biology. Specifically, these miRNAs exhibit high prognostic significance in lung, cervical, and liver cancer, as suggested by their involvement in several cancer-related pathways. Significance StatementWe highlight an undescribed non-canonical role of the mammalian Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APE1) in the context of RNA G-quadruplexes (rG4), specifically in the alternative pathway of miRNA maturation of guanosine-rich miRNA precursors. Specifically, APE1 binds these structures and modulates their folding, mainly through its N-terminal region and some residues in its catalytic domain. Moreover, we showed an interesting new role of APE1 in regulating the shuttling and accumulation of miR-92b between the nuclear and cytosolic compartments, opening new perspectives on how APE1 may exercise its role in the miRNA maturation pathway and function. Moreover, APE1-depleted dysregulated miRNAs with rG4 motifs in their precursors have significant prognostic value in lung, cervical, and liver tumors, suggesting potential targets for cancer therapy.

We present a comprehensive, experimental and theoretical study of the impact of 5-hydroxymethylation of DNA cytosine. Using molecular dynamics, biophysical experiments and NMR spectroscopy, we found that Ten-Eleven translocation (TET) dioxygenases generate an epigenetic variant with structural and physical properties not too different to those of 5-methylcytosine. Experiments and simulations demonstrate that 5-methyl-cytosine (mC) and 5-hydroxymethyl-cytosine (hmC) generally lead to more rigid duplexes with poorer circularization efficiencies and lower ability to form nucleosomes. In particular, we can rule out the hypothesis that hydroxymethylation reverts to unmodified cytosine physical properties, as hmC is even more rigid than mC. Thus, we do not expect dramatic changes in the chromatin structure induced by differences in physical properties between d(mCpG) and d(hmCpG). On the contrary, our simulations suggest that methylated-DNA binding domains (MBD), associated with repression activities, are very sensitive to the substitution d(mCpG)[->] d(hmCpG), while MBD3 which has a dual activation/repression activity is not sensitive to the d(mCpG) [->] d(hmCpG) change. Overall, while changes in gene activity due to cytosine methylation are the result of the combination of stiffness-related chromatin reorganization and MBD binding, those associated to 5-hydroxylation of methylcytosine could be explained by a change in the balance of repression/activation pathways related to differential MBD binding.

To understand how genes are regulated it is important to assess the molecular basis of TFs cooperation in promoter and enhancer regulatory elements. Compelling genetic, genomic and biochemical data in mammals and zebrafish point at DECA-CCAAT composite elements as important for regulation of gene expression in development. DECA is recognized by the homeodomain (HD) heterodimeric TALE TFs (PBX/PREP), CCAAT by the NF-Y trimer. Both are evolutionarily conserved in eukaryotes, including plants. Sp2, a member of the Sp/KLF family, potentiates association of TALE and NF-Y on DNA. We applied AlphaFold to infer the structural basis of the hexameric complex with DNA. The resulting models position TALE and NF-Y on the respective sites, predicting the complex arrangement of the MEINOX/PBC heterodimerization module of the TALE TFs and the sequence-specific DNA contacts of the HDs to DECA. The complex is tied by the Sp2 N-terminus, which contacts the TALE dimer through the -helical SP-box and the NF-Y trimer through a novel short linear motif (YA-SLiM). A flexible linker separates the anchor points, consistent with the constrained stereo-alignment of the DECA-CCAAT motif. The models are confirmed by assembly of the recombinant NF-Y/TALE/Sp2 in complex with DNA in vitro. Mutagenesis confirms the importance of the SP-box and YA-SLiM for cooperativity. Rationalising available genomic and biochemical data, the structural models depict a novel mode of cooperative binding on DNA, providing important clues as to how TFs potentially function beyond DECA-CCAAT in different eukaryotic contexts.

Bacteriophage mv4 is a temperate bacterial virus able to integrate its genome at the 3 end of the tRNASER of Lactobacillus delbrueckii subsp. bulgaricus chromosome through site-specific recombination. Previous investigations revealed that the mv4Int/attP/attB recombination module was atypical compared to conventional heterobivalent tyrosine recombinases, such as the paradigmatic Lambdavirus lambda integrase, suggesting alternative recombination mechanism. In vitro recombination assays with random DNA libraries were used to comprehensively delineate the mv4 recombination system. We showed that mv4Int is a 369-aa protein that exhibits all structural hallmarks of integrases from the Tn916 family and interacts cooperatively with its recombination sites. We established that mv4Int distinguishes itself from classical heterobivalent integrases by a greater tolerance to nucleotide variations in attB and core-attP sites. We demonstrated that, upon considering nucleotide degeneracy, the 21-bp core-attP and attB recombination sites share structural similarities with classical heterobivalent integrase systems, with two 7-bp inverted-repeat regions corresponding to mv4Int core-binding sites surrounding a 7-bp strand-exchange region. Furthermore, our study highlighted compositional biases and nucleotide interdependencies within the core-binding regions that exerted a significant influence on the outcomes of recombination events.

Pioneer transcription factors (PTFs) have the remarkable ability to directly bind to chromatin for stimulating vital cellular processes. In this work, we dissect the universal binding mode of Sox PTF by combining extensive molecular simulations and DNA footprinting techniques. As a result, we show that when Sox consensus DNA is located at the solvent-facing DNA strand, Sox binds to the compact nucleosome without imposing any significant conformational changes. We also reveal that the basespecific Sox:DNA interactions (base reading) and the Sox-induced DNA changes (shape reading) are concurrently required for the sequence-specific DNA recognition. Among different nucleosomal positions, such a specific reading mechanism is satisfied solely at superhelical location 2 (SHL2). While SHL2 acts transparently to Sox binding, SHL4 permits only shape reading, and SHL0 (dyad) allows no reading mechanism. These findings demonstrate for the first time that Sox-based nucleosome recognition is essentially guided by the distinct histone-DNA interactions, permitting varying degrees of DNA flexibility.

Previous studies indicate that genomic loci harboring G-quadruplexes (G4s)--stacked structures that can form in single-stranded DNA--can be linked to epigenetic instability. However, the role of chromatin redistribution and the genome-wide nature of this process need further investigation. Here, we provide experimental evidence that connects G4s to alterations in the deposition of chromatin proteins. We have used metabolic labelling and immunoprecipitation of new and parental proteins in hRPE-1 cells to investigate global chromatin deposition dynamics. We identify a reciprocal, local bias in chromatin protein deposition at G4 sites favoring the association of parental proteins with the G4 and new proteins with the C4 DNA strand. The deposition bias at G4 sites does not depend on replication directionality and is strengthened by G4 stabilization. Slowing down replication forks upon hydroxyurea treatment reverses the bias, supposedly affected by decoupling between helicase and polymerase. Interestingly, upon combined G4 stabilization and slowing of the replication forks, new proteins exhibit a redistribution pattern similar to G4 stabilization alone, while parental protein redistribution more resembles one after hydroxyurea treatment, hinting at mechanistic differences between parental and new histone distribution. We also report that the genomic distribution of putative quadruplexes is not random and depends on loop size, where G4s with shorter loops have a preference for the DNA strand replicated by leading and G4s with longer loops by lagging strand replication. These findings provide insight into the mechanisms behind G4 occurrence and its role in epigenetic instability and help to improve our understanding of the factors influencing biases in global chromatin protein redeposition.

G-quadruplex (G4) structures are critical regulators of gene expression, yet the role of an individual G4 within its native chromatin remains underexplored. Here, we used CRISPR-Cas9 to introduce guanine-to-thymine mutations at a G4-forming motif within the adh1+ promoter in yeast, creating two mutant strains: one with G4-only mutations and another with both G4 and TATA-box mutations. Chromatin immunoprecipitation using BG4 antibody confirmed reduced G4 enrichment in both mutants, validating G4 structure formation in the wild-type chromatin. Detailed characterizations demonstrated that the G4 mutations alter its dynamics without fully preventing its formation. These mutations significantly reduce adh1 transcript levels, with G4 TATA-box mutant causing the strongest transcriptional suppression. This indicates a positive regulatory role for the Adh1 G4 structure in adh1+ gene expression. Furthermore, both mutants displayed altered transcriptomic profiles, particularly impacting the oxidoreductase pathway. Metabolomic analyses by mass spectrometry further highlighted substantial disruptions in NAD+/NADH metabolism, a key energy reservoir for metabolic regulation. Together, our findings illustrate how deregulation of a single G4 structure influences transcriptome regulation, with implications for metabolic diseases. It also highlights the therapeutic potential of G4 modulation as a novel, controlled approach to reprogram cellular metabolism to achieve targeted phenotypic shifts. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/643214v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@10262a6org.highwire.dtl.DTLVardef@19aa1a2org.highwire.dtl.DTLVardef@e99487org.highwire.dtl.DTLVardef@11639e5_HPS_FORMAT_FIGEXP M_FIG C_FIG

The signaling molecule auxin is pivotal in coordinating many growth and development processes in plants mainly through the modulation of gene expression. The transcriptional response to auxin is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif (TGTC[TC]/[GG]) called the auxin-response element (AuxRE). ARFs can homodimerize through their DNA binding domains (DBD) thereby enabling cooperative binding for a bipartite inverted AuxRE (IR7). In addition to the DBD, most ARFs contain a C-terminal Phox and Bem1p (PB1) domain both capable of homotypic interactions, and mediating interactions with Aux/IAA repressors. Given the dual role of the PB1 domain, and the ability of both DBD and PB1 domain to mediate dimerization, a key question is how each of these domains contributes to conferring DNA-binding specificity and affinity. So far, ARF-ARF and ARF-DNA interactions have mostly been approached using qualitative methods that do not provide a quantitative and dynamic view on the binding equilibria. Here, we utilize a DNA binding assay based on single-molecule Forster resonance energy transfer (smFRET) to study the affinity and kinetics of the interaction of several Arabidopsis thaliana ARFs with an IR7 AuxRE. We show that both DBD and PB1 domains of AtARF2 contribute toward DNA binding, and we identify ARF dimer stability as a key parameter in defining affinity and kinetics seen for the DBDs of different AtARFs. Lastly, we derived an analytical solution for a four-state cyclic model that explains both the kinetics and the affinity of the interaction between AtARF2 and IR7. Our work demonstrates that the affinity of ARFs towards composite DNA response elements can be tuned by small changes of their dimerization equilibrium suggesting that this effect has major implications for ARF-mediated transcriptional activity.

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.