Biology subjects

Hickman, A. B.

Publications and source records attributed to Hickman, A. B..

4 recordsLinked to original sources

bioRxiv2026

Structure-based recasting of a mammalian DNA transpososomeas an obligate heterodimer

Eukaryotic DNA transpososomes assemble as nucleoprotein complexes containing multiple identical transposase protomers. We determined the structure of the hyperactive Myotis lucifugus piggyBat transpososome and discovered that it uses an unusual crescent-shaped, asymmetric tetramer to synapse divergent inverted terminal repeats. We found that identical amino-acid sequence motifs adopt distinct roles to mediate two modes of DNA binding: one to perform strand transfer and one, devoid of catalytic activity, that promotes synapsis while simultaneously protecting the transposon from auto-destructive internal cleavage by its active sites. Guided by the observed modularity of the assembly, we engineered an obligate heterodimeric system by identifying mutations that suppress homodimer formation and paired this with specific point mutations that prevent non-targeted integration. By adding to the heterodimer two different TALE domains designed to bind a human genomic safe harbor sequence, we achieved >98% targeted integration at the intended sequence in a plasmid-based assay, validating the viability of heterodimeric transposases for genomic applications.

molecular biology↗

bioRxiv2024

Molecular basis for the transcriptional regulation of an epoxide-based virulence circuit in Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa infects cystic fibrosis (CF) patient airways and produces a virulence factor Cif that is associated with worse outcomes. Cif is an epoxide hydrolase that reduces cell-surface abundance of the cystic fibrosis transmembrane conductance regulator (CFTR) and sabotages pro-resolving signals. Its expression is regulated by a divergently transcribed TetR family transcriptional repressor. CifR represents the first reported epoxide-sensing bacterial transcriptional regulator, but neither its interaction with cognate operator sequences nor the mechanism of activation has been investigated. Using biochemical and structural approaches, we uncovered the molecular mechanisms controlling this complex virulence operon. We present here the first molecular structures of CifR alone and in complex with operator DNA, resolved in a single crystal lattice. Significant conformational changes between these two structures suggest how CifR regulates the expression of the virulence gene cif. Interactions between the N-terminal extension of CifR with the DNA minor groove of the operator play a significant role in the operator recognition of CifR. We also determined that cysteine residue Cys107 is critical for epoxide sensing and DNA release. These results offer new insights into the stereochemical regulation of an epoxide-based virulence circuit in a critically important clinical pathogen.

biochemistry↗

bioRxiv2023

Toxic anti-phage defense proteins inhibited by intragenic antitoxin proteins

Recombination-promoting nuclease (Rpn) proteins are broadly distributed across bacterial phyla, yet their functions remain unclear. Here we report these proteins are new toxin-antitoxin systems, comprised of genes-within-genes, that combat phage infection. We show the small, highly variable Rpn C-terminal domains (RpnS), which are translated separately from the full-length proteins (RpnL), directly block the activities of the toxic full-length proteins. The crystal structure of RpnAS revealed a dimerization interface encompassing a helix that can have four amino acid repeats whose number varies widely among strains of the same species. Consistent with strong selection for the variation, we document plasmid-encoded RpnP2L protects Escherichia coli against certain phages. We propose many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms. SignificanceHere we document the function of small genes-within-genes, showing they encode antitoxin proteins that block the functions of the toxic DNA endonuclease proteins encoded by the longer rpn genes. Intriguingly, a sequence present in both long and short protein shows extensive variation in the number of four amino acid repeats. Consistent with a strong selection for the variation, we provide evidence that the Rpn proteins represent a phage defense system.

microbiology↗

bioRxiv2022

Formation of the active Hermes transpososome is driven by asymmetric DNA binding of BED domains

The cut-and-paste Hermes DNA transposase stands out among the transposases that have been biochemically or structurally characterized so far. Many transposases function as dimers, but the Hermes transposase forms a tetramer of dimers to achieve its active form in vivo. Intriguingly, the transposition complex, or transpososome, relies on only one dimer to perform the enzymatic reactions necessary to the mobilization of its transposon. Our investigation combining biochemical and structural approaches shows that the Hermes octamer extensively interacts with its transposon left-end (LE) engaging the BED domains of three Hermes protomers belonging to three dimers. By contrast, the right-end (RE) is entirely deprived of such interaction inside the transpososome. Our work suggests that formation of the Hermes synaptic complex is sequential and relies on the considerable difference of affinity of the transposase towards its transposon ends. Thus, we propose that Hermes dimers multimerize to gather enough BED domains to find the LE among the abundant genomic DNA, facilitating the subsequent interaction with the RE, most likely solely based on recognition of its terminal inverted repeat.

biochemistry↗