Biology subjects

Doyle, L. A.

Publications and source records attributed to Doyle, L. A..

6 recordsLinked to original sources

bioRxiv2026

The activity, divergence, and evolutionary degradation of modern-day homing endonucleases and their reconstructed ancestors

Homing endonucleases (HEs) are selfish genetic elements that drive the mobilization of their own coding sequences, often in concert with surrounding introns. Homing endonuclease genes (HEGs) usually display life cycles in which they accumulate inactivating mutations after invading a host genomic target site, leading to eventual removal from the genome. We identified several hundred novel HEGs and determined the distribution of their proteins behaviors and activities. Approximately 10% are expressed as properly folded functional proteins that cleave predictable DNA target sites. Another [~]20% display significant expression but little to no cleavage activity; the remainder display severely reduced expression. Despite the presence of debilitating mutations throughout most HEGs, ancestral reconstructions yielded endonucleases with improved expression and stability. One such reconstruction, at a hypothetical node preceding highly diverged HEs that cleave unique target sites, binds (but does not cleave) their individual targets. It instead cleaves a DNA sequence that represents a hybrid of those modern-day DNA targets, while displaying a specificity profile that resembled those of previously characterized HEs. Its DNA-bound crystal structure adds detail to our understanding of how homing endonuclease DNA contacting surfaces and residues shift and rearrange during evolution, ultimately leading to their action at new target sites.

biochemistry↗

bioRxiv2026

Competing forms of protein-protein association and DNA binding exhibited by BrxC from the BREX phage restriction system

Bacteriophage exclusion (BREX) defense systems restrict phage infection via inhibition of phage DNA replication, while also modifying and protecting the bacterial genome. Type I BREX systems encode six conserved proteins, including a site-specific DNA methyltransferase. Host methylation requires a subset of BREX proteins, whereas phage restriction generally requires them all, suggesting that distinct but overlapping complexes mediate these activities. Full details of the mechanism and regulation of BREX remains to be understood. Here, we characterize the behavior and structures of the conserved BrxC AAA+ ATPase protein. BrxC forms multiple competing assemblages - various self-associating multimers, as well as a complex with BrxB-PglZ - that can be uncoupled via distinct point mutations, leading to differing effects on host methylation versus phage restriction. BrxCs self-association, as well as its ability to bind DNA, is regulated by ATP binding and hydrolysis; BrxA and BrxB appear to also regulate those behaviors. These collective results suggest that BrxC may play a key role in controlling the two activities of BREX, with BrxB, BrxC and PglZ forming a core complex, and the equilibrium among competing assemblies containing those proteins modulating the balance between idling and activated restrictive states.

biochemistry↗

bioRxiv2025

H2AK119ub Safeguards Against Ectopic Transcription Factor Mediated Gene Activation in the Developing Forebrain

The PRC1 complex regulates developmental gene expression in mammals by ubiquitinating histones (H2AK119ub) and nucleating repressive chromatin interactions. Human genetic data and functional experimentation have demonstrated that H2AK119ub is required for cortical development, however the molecular mechanism for this remains unknown. Here, we show that mouse embryos expressing catalytically deficient RING1B exhibit intact early neurogenesis but display impaired neuroectodermal fate restriction. Using in vivo, primary, and in vitro models, we demonstrate that reduced H2AK119ub leads to ectopic activation of lineage-inappropriate transcriptional programmes, including mesodermal and endodermal gene expression during neural differentiation. This transcriptional deregulation is not solely attributable to H2AK119ub or H3K27me3 loss but instead reflects sensitisation of PRC1 target genes to inappropriate transcription factor (TF)-mediated activation. Synthetic induction of candidate TFs, including GATA6, SOX7, and SNAI1, phenocopies the fate-skewing effects of PRC1 catalytic dysfunction, confirming their causal role. Our results uncover a buffering role for PRC1-catalysed H2AK119ub in safeguarding neural progenitor identity by preventing inappropriate TF-driven transcription and provides a mechanistic framework for understanding the cellular heterogeneity and phenotypic variability observed in Polycomb-associated neurodevelopmental disorders.

molecular biology↗

bioRxiv2023

Computational design of sequence-specific DNA-binding proteins

Sequence-specific DNA-binding proteins (DBPs) play critical roles in biology and biotechnology, and there has been considerable interest in the engineering of DBPs with new or altered specificities for genome editing and other applications. While there has been some success in reprogramming naturally occurring DBPs using selection methods, the computational design of new DBPs that recognize arbitrary target sites remains an outstanding challenge. We describe a computational method for the design of small DBPs that recognize specific target sequences through interactions with bases in the major groove, and employ this method in conjunction with experimental screening to generate binders for 5 distinct DNA targets. These binders exhibit specificity closely matching the computational models for the target DNA sequences at as many as 6 base positions and affinities as low as 30-100 nM. The crystal structure of a designed DBP-target site complex is in close agreement with the design model, highlighting the accuracy of the design method. The designed DBPs function in both Escherichia coli and mammalian cells to repress and activate transcription of neighboring genes. Our method is a substantial step towards a general route to small and hence readily deliverable sequence-specific DBPs for gene regulation and editing.

synthetic biology↗

bioRxiv2021

Identification and characterization of BrxR as a regulatory gene in the BREX phage restriction system

Bacteriophage exclusion ( BREX) phage restriction systems are found in a wide range of bacteria. Various BREX systems encode unique combinations of proteins that usually include a site-specific methyltransferase; none appear to contain a nuclease. Here we describe the identification and characterization of a Type I BREX system from Acinetobacter and the effect of deleting each BREX ORF on growth, methylation, and restriction. We identified a previously uncharacterized gene in the BREX operon that is dispensable for methylation but involved in restriction. Biochemical and crystallographic analyses of this factor, which we term BrxR ( BREX Regulator), demonstrate that it forms a homodimer and specifically binds a DNA target site upstream of its transcription start site. Deletion of the BrxR gene causes cell toxicity, reduces restriction, and significantly increases the expression of BrxC. In contrast, the introduction of a premature stop codon into the BrxR gene, or a point mutation blocking its DNA binding ability, has little effect on restriction, implying that the BrxR coding sequence and BrxR protein play independent functional roles. We speculate that the BrxR coding sequence is involved in cis regulation of anti-phage activity, while the BrxR protein plays an additional regulatory role, perhaps during horizontal transfer.

molecular biology↗

bioRxiv2021

The structural basis of mRNA recognition and binding by eukaryotic pseudouridine synthase PUS1

The chemical modification of RNA bases represents a ubiquitous activity that spans all domains of life. Pseudouridylation is the most common RNA modification and is observed within tRNA, rRNA, ncRNA and mRNAs. Pseudouridine synthase or PUS enzymes include those that rely on guide RNA molecules and others that function as stand-alone enzymes. Among the latter, several have been shown to modify mRNA transcripts. Although recent studies have defined the structural requirements for RNA to act as a PUS target, the mechanisms by which PUS1 recognizes these target sequences in mRNA are not well understood. Here we describe the crystal structure of yeast PUS1 bound to an RNA target that we identified as being a hot spot for PUS1-interaction within a model mRNA at 2.4 [A] resolution. The enzyme recognizes and binds both strands in a helical base-paired RNA duplex, and thus guides the RNA containing the target uridine to the active site for subsequent modification of the transcript. The study also allows us to show the divergence of related PUS1 enzymes and their corresponding RNA target specificities, and to speculate on the basis by which PUS1 binds and modifies mRNA or tRNA substrates.

biophysics↗