Biology subjects

Montgomery, J. S.

Publications and source records attributed to Montgomery, J. S..

8 recordsLinked to original sources

bioRxiv2026

Enhanced DNA Scanning and Target Specificity via Motif Sequestration

Sequence-specific DNA-binding proteins must efficiently locate their target sequences within large, structurally heterogeneous genomes while avoiding nonproductive off-target interactions. Here, we show that an autoinhibitory conformational state of the site-specific recombinase Cre lowers nonspecific DNA-binding affinity to promote one-dimensional DNA scanning while suppressing off-target assembly of recombinogenic complexes. Recent studies indicate that the C-terminal segment of Cre transiently adopts a sequestered conformation that occludes the catalytic domain's DNA-binding surface. Using NMR spectroscopy, single-molecule fluorescence microscopy, and optical trapping, we investigated how this conformational equilibrium regulates target search. NMR measurements reveal that the sequestered state predominates in the absence of DNA and persists at noncognate sites, whereas binding to cognate loxP sequences relieves autoinhibition. Single-molecule tracking demonstrates that full-length Cre undergoes processive one-dimensional diffusion and transitions to stable binding upon cognate-site recognition, whereas C-terminal truncation impairs scanning efficiency by arresting diffusion. These findings demonstrate how transient motif sequestration coordinates target search with site-specific complex assembly, suggesting a general mechanism by which DNA-modifying enzymes balance search efficiency and target specificity.

biophysics↗

bioRxiv2026

Thermodynamics of Indirect Readout in Cre-loxP Recognition

Cre, a site-specific tyrosine DNA recombinase, enables targeted manipulation of genetic material without production of cytotoxic double-strand DNA breaks. Though widely deployed in biotechnology, an incomplete understanding of how Cre recognizes its cognate loxP target limits its broader application in human health. Cre has been proposed to recognize an inverted pair of recombinase binding elements (RBEs) at loxP sites using an indirect readout mechanism, inducing conformations that are disfavored at noncognate sites. Despite a high degree of specificity, structural studies have shown that Cre protomers make few direct base-specific contacts to each RBE, implicating noncontacted positions in recognition by tuning flexibility and enabling binding-coupled conformational changes. We designed a set of loxP half-site variants predicted to rigidify the DNA substrate and measured the thermodynamics of Cre binding by isothermal titration calorimetry. Thermodynamic signatures and NMR spectra reveal that unfavorable mutations at noncontacted positions in the RBE reduce binding- coupled conformational changes. Moreover, mass photometry of Cre binding to oligonucleotides containing two properly spaced RBEs revealed that non-contacted positions in the intervening 8-base pair spacer influence cooperative dimerization of two Cre protomers at loxP sites, and their synapsis to form catalytically active Cre4-loxP2 complexes. These results demonstrate that noncontacted positions contribute to specificity by encoding favorable DNA mechanics, offering new design principles for engineering Cre variants that target alternative DNA sequences.

biophysics↗

bioRxiv2024

Protein and DNA Conformational Changes Contribute to Specificity of Cre Recombinase

Cre, a conservative site-specific tyrosine recombinase, is a powerful gene editing tool in the laboratory. Expanded applications in human health are hindered by lack of understanding of the mechanism by which Cre selectively binds and recombines its cognate loxP sequences. This knowledge is essential for retargeting the enzyme to new sites and for mitigating effects of off-target recombination. Prior studies have suggested that in addition to a few base-specific contacts to cognate loxP DNA, the enzymes specificity is enhanced by (1) autoinhibition involving a conformational change in the proteins C-terminal helix, and (2) indirect readout via binding-coupled conformational changes in the target DNA. We used isothermal titration calorimetry (ITC), circular dichroism (CD) and heteronuclear NMR spectroscopy to investigate DNA site recognition by wild-type Cre and a deletion mutant lacking the C-terminal helix. ITC of Cre and a C-terminal deletion variant against cognate and non-cognate DNA recombinase binding elements (RBEs) reveal that the C-terminus reduces DNA binding affinity by six-fold towards cognate DNA. Additionally, ITC revealed highly unfavorable binding enthalpy, which when combined with evidence from CD and NMR of structural differences between cognate and non-cognate complexes support a model in which binding-coupled DNA bending provides a unique structure-thermodynamic signature of cognate complexes. Together, these findings advance our understanding of site-recognition by Cre recombinase. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/627928v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@19fb34dorg.highwire.dtl.DTLVardef@1b4b025org.highwire.dtl.DTLVardef@1988254org.highwire.dtl.DTLVardef@1945cb1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Chromosome-level assemblies of Amaranthus palmeri, Amaranthus retroflexus, and Amaranthus hybridus allow for genomic comparisons and identification of a sex-determining region

Amaranthus palmeri (Palmer amaranth), A. retroflexus (redroot pigweed), and A. hybridus (smooth pigweed) are troublesome weeds that are economically damaging to several cropping systems. Collectively referred to as pigweeds, these species are incredibly adaptive and have become successful competitors in diverse agricultural settings. Development of genomic resources for these species promises to facilitate the elucidation of the genetic basis of traits such as biotic and abiotic stress tolerance (e.g., herbicide resistance) and sex determination. Here, we sequenced and assembled chromosome-level genomes of these three pigweed weed species. By combining the haplotype-resolved assembly of A. palmeri with existing restriction site-associated DNA sequencing data, we identified a [~]2.84 Mb region on chromosome 3 of Hap1 that is male-specific and contains 37 genes. Transcriptomic analysis revealed two genes within the male-specific region, RESTORER OF FERTILITY 1 (Rf1) and TLC DOMAIN-CONTAINING PROTEIN (TLC), were upregulated in male individuals across the shoot apical meristem, the floral meristem, and mature flowers, indicating their potential involvement in sex determination in A. palmeri. In addition, we rigorously classified cytochrome P450 genes in all three pigweeds due to their involvement in non-target site herbicide resistance. Finally, we identified contiguous extrachromosomal circular DNA (eccDNA) in A. palmeri, a critical component of glyphosate resistance in this species. The findings of this study advance our understanding of sex determination in A. palmeri and provide genomic resources for elucidating the genetic basis and evolutionary origins of adaptive traits within the genus.

genomics↗

bioRxiv2024

A transposable element insertion in IAA16 interrupts normal splicing and generates a novel dicamba resistance allele in Bassia scoparia

A dicamba-resistant population of kochia (Bassia scoparia) identified in Colorado, USA in 2012 was used to generate a synthetic mapping population that segregated for dicamba resistance. Linkage mapping associating dicamba injury with genotype derived from restriction-site-associated DNA sequencing identified a single locus in the kochia genome associated with resistance on chromosome 4. A mutant version of Auxin/Indole-3-Acetic Acid 16 (AUX/IAA16; a gene previously implicated in dicamba resistance in kochia) was found near the middle of this locus in resistant plants. Long read sequencing of dicamba-resistant plants identified a recently inserted Ty1/Copia retrotransposon near the beginning of the second exon of AUX/IAA16, leading to disruption of normal splicing. A molecular marker for this insertion allows for rapid detection of resistance. Stable transgenic lines of Arabidopsis thaliana ectopically expressing the mutant and wildtype alleles of AUX/IAA16 were developed. Arabidopsis thaliana plants expressing the mutant AUX/IAA16 allele grew shorter roots on control media. However, transgenic root growth was less inhibited on media containing either dicamba (5 M) or IAA (0.5 M) when compared to non-transgenic plants or those expressing the wildtype allele of AUX/IAA16. In vitro assays indicate reduced binding affinity and more rapid dissociation of the mutant AUX/IAA with TIR1 in the presence of several auxins, and protein modeling suggests the substitution of the glycine residue in the degron domain of AUX/IAA16 is especially important for resistance. A fitness cost associated with the mutant allele of AUX/IAA16 has implications for resistance evolution and management of kochia populations with this resistance mechanism. SignificanceAuxin mimics are amongst the most important herbicides in modern agriculture. Evolution of weeds that are resistant to these herbicides threatens sustainable crop production. Understanding the basis of auxin herbicide resistance informs the development of improved weed control technologies. Additionally, auxin-resistant mutations and their pleotropic effects help us understand auxin perception and signalling. We describe a transposable element insertion within an herbicide target site gene that alters splicing and reduces synthetic and natural auxin perception.

plant biology↗

bioRxiv2024

A phased chromosome-level genome assembly provides insights into the evolution of sex chromosomes in Amaranthus tuberculatus

O_LIAmaranthus tuberculatus (waterhemp) is a troublesome weed species of agronomic importance that is dioecious with an XY sex-determination system. The evolution of sex chromosomes, the contiguity of sex-determining region (SDR) and the expression pattern of genes within the SDR remain poorly understood. C_LIO_LIWe assembled the genome of a male A. tuberculatus, phased the genome into two chromosome-level haplotypes, and performed restriction site-associated DNA genome- wide association (RAD-GWA) analysis, comparative genomics, adaptive evolution analysis, and, with existing data, transcriptomic profiling to characterize the species sex chromosomes. C_LIO_LIComparative analysis enabled the identification of a [~]32.8 Mb SDR on chromosome 1 that is gene-poor, abundant in long terminal repeat (LTR) retrotransposons, and harbors two inversions. Synteny analysis revealed that chromosome 1 likely originated from the fusion of two ancestral chromosomes, and mRNA data indicated 93 genes out of the 531 protein-coding genes within the SDR of haplome 2 were differentially expressed between mature male and female flowers, with several of the genes enriched for Gene Ontology (GO) terms involved in floral development. C_LIO_LIBeyond adding to our understanding of sex chromosome evolution, the genomic resource provided here will be valuable for addressing further questions on adaptive trait evolution in Amaranthus. C_LI

genomics↗

bioRxiv2023

The International Weed Genomics Consortium: Community Resources for Weed Genomics Research

The International Weed Genomics Consortium is a collaborative group of researchers focused on developing genomic resources for the study of weedy plants. Weeds are attractive systems for basic and applied research due to their impacts on agricultural systems and capacity to swiftly adapt in response to anthropogenic selection pressures. Our goal is to use genomic information to develop sustainable and effective weed control methods and to provide insights about biotic and abiotic stress tolerance to assist crop breeding. Here, we outline resources under development by the consortium and highlight areas of research that will be impacted by these enabling resources.

genomics↗

bioRxiv2023

Complexes of vertebrate TMC1/2 and CIB2/3 proteins form hair-cell mechanotransduction cation channels

Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of the inner-ear mechano-electrical transduction (MET) apparatus. These interactions have been proposed to be functionally relevant across mechanosensory organs and vertebrate species. Here we show that both CIB2 and CIB3 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET function in mouse cochlea and vestibular end organs as well as in zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that vertebrate CIB proteins can simultaneously interact with at least two cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3. Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict that TMCs are structurally stabilized by CIB proteins to form cation channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2 complexes are integral to hair-cell MET function in vertebrate mechanosensory epithelia.

neuroscience↗