Biology subjects

Fisher, G.

Publications and source records attributed to Fisher, G..

6 recordsLinked to original sources

bioRxiv2023

Substrate accessibility regulation of human TopIIa decatenation by cohesin

Human topoisomerase II alpha (TOP2) is the main mitotic decatenase 1-3 resolving intertwines between sister chromatids that form during DNA replication 4. Here, we employ quadruple-trap optical tweezers to generate braids between a pair of {lambda}-DNA molecules to study, at the single-molecule level and in real-time, how TOP2 untangles these DNAs. We find that TOP2 rapidly resolves single and multiple braids and is inhibited by chemotherapy agent, etoposide. TOP2 is sensitive to DNA conformation, exhibiting a chiral preference for the removal of braids with right-handed crossings and is inhibited when DNAs are held at forces over 20pN. We show that TOP2 must load at the cross between the two DNAs to resolve braids efficiently. TOP2 pre-loaded onto individual DNAs is unable to resolve newly formed braids in the presence of ATP, suggesting that the geometry of pre-bound enzyme is incompatible with the capture of the second DNA. Finally, we show that cohesin binds to braids, preventing TOP2 from resolving them. Our study unveils novel insights into the regulation of TOP2s decatenation, underscoring the importance of substrate accessibility and the role of cohesin in modulating TOP2 activity.

biophysics↗

bioRxiv2021

Structure, dynamics, and inhibition of Staphylococcus aureus m1A22-tRNA methyltransferase

The enzyme m1A22-tRNA methyltransferase (TrmK) catalyses the transfer of a methyl group from SAM to the N1 of adenine 22 in tRNAs. TrmK is essential for Staphylococcus aureus survival during infection, but has no homologue in mammals, making it a promising target for antibiotic development. Here we describe the structural and functional characterisation of S. aureus TrmK. Crystal structures are reported for S. aureus TrmK apoenzyme and in complexes with SAM and SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favourable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated by a large favourable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. Activity assays for S. aureus TrmK-catalysed methylation of WT and position 22 mutants of tRNALeu demonstrate that the enzyme requires an adenine at position 22 of the tRNA. Intriguingly, a small RNA hairpin of 18 nucleotides is methylated by TrmK depending on the position of the adenine. In-silico screening of compounds suggested plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92, in the vicinity of the SAM-binding site, as the sole residue modified. These results these results identify a cryptic binding pocket of S. aureus TrmK and lay the foundation for future structure-based drug discovery.

biochemistry↗

bioRxiv2021

Competitive binding of MatP and topoisomerase IV to the MukB dimerization hinge

SMC complexes have ubiquitous roles in chromosome organisation. In Escherichia coli, the interplay between the SMC complex, MukBEF, and matS-bound MatP in the replication termination region, ter, results in depletion of MukBEF from ter, thus promoting chromosome individualisation by directing replichores to separate cell halves. MukBEF also interacts with topoisomerase IV ParC2E2 heterotetramers, to direct its chromosomal distribution to mirror that of MukBEF, thereby facilitating coordination between chromosome organisation and decatenation by topoisomerase IV. Here we demonstrate that the MukB dimerization hinge binds ParC and MatP with the same dimer to dimer stoichiometry. MatP and ParC have an overlapping binding interface on the MukB hinge, leading to their mutually exclusive binding. Furthermore, the MukB hinge fails to stably associate with matS-bound MatP, while MatP mutants deficient in matS binding are impaired in MukB hinge binding, demonstrating that mats competes with the hinge for MatP binding. Cells expressing MukBEF complexes containing a mutation in the MukB hinge interface for ParC/MatP binding are deficient in ParC binding in vivo, despite having a Muk+ topoisomerase IV+ phenotype. This mutant protein is also impaired in MatP binding in vitro, and cells expressing this variant exhibit a MukBEF cellular localisation consistent with impaired MatP binding.

biochemistry↗

bioRxiv2020

An evolutionary model identifies the main selective pressures for the evolution of genome-replication profiles.

Recent results comparing the temporal program of genome replication of yeast species belonging to the Lachancea clade support the scenario that the evolution of replication timing program could be mainly driven by correlated acquisition and loss events of active replication origins. Using these results as a benchmark, we develop an evolutionary model defined as birth-death process for replication origins, and use it to identify the selective pressures that shape the replication timing profiles. Comparing different evolutionary models with data, we find that replication origin birth and death events are mainly driven by two evolutionary pressures, the first imposes that events leading to higher double-stall probability of replication forks are penalized, while the second makes less efficient origins more prone to evolutionary loss. This analysis provides an empirically grounded predictive framework for quantitative evolutionary studies of the replication timing program.

evolutionary biology↗

bioRxiv2019

Unilateral neglect is associated with poor proprioception after stroke - a systematic review

BackgroundProprioceptive impairment is a potential contributing factor to the clinical presentation of Unilateral Neglect (UN), a common and debilitating condition that can occur after stroke. To date there has not been a comprehensive review of studies examining the various aspects of proprioception in UN after stroke.\n\nAimTo determine if the presence of UN is associated with more severe proprioceptive deficit in stroke affected populations.\n\nMethodsThe MEDLINE, Embase, Scopus, CINAHL and Web of Science databases were searched from inception to January 2019 using an a priori search strategy. Two independent reviewers screened abstracts and full texts. Two reviewers then independently extracted data from each full text. A third reviewer resolved disagreements at each step. Risk of bias was assessed using the AXIS Quality Assessment tool. For full protocol see PROSPERO, registration number CRD42018086070. One-hundred and sixty-seven abstracts were identified, of which fifty-four were eligible for full text screening. A total of 18 papers were included in the review.\n\nConclusionsMore severe proprioceptive deficit is associated with the presence of UN after stroke. However, the available evidence is limited by the large heterogeneity of assessment of both UN and proprioception, and level of study quality. UN and proprioception are seldom completely assessed in research, and it is likely this is true in everyday clinical practice.

neuroscience↗

bioRxiv2017

Deciphering microbial interactions in synthetic human gut microbiome communities

The human gut microbiota comprises a dynamic ecological system that contributes significantly to human health and disease. The ecological forces that govern community assembly and stability in the gut microbiota remain unresolved. We developed a generalizable model-guided framework to predict higher-order consortia from time-resolved measurements of lower-order assemblages. This method was employed to decipher microbial interactions in a diverse 12-member human gut microbiome synthetic community. We show that microbial growth parameters and pairwise interactions are the major drivers of multi-species community dynamics, as opposed to context-dependent (conditional) interactions. The inferred microbial interaction network as well as a top-down approach to community assembly pinpointed both ecological driver and responsive species that were significantly modulated by microbial inter-relationships. Our model demonstrated that negative pairwise interactions could generate history-dependent responses of initial species proportions on physiological timescales that frequently does not originate from bistability. The model elucidated a topology for robust coexistence in pairwise assemblages consisting of a negative feedback loop that balances disparities in monospecies fitness levels. Bayesian statistical methods were used to evaluate the constraint of model parameters by the experimental data. Measurements of extracellular metabolites illuminated the metabolic capabilities of monospecies and potential molecular basis for competitive and cooperative interactions in the community. However, these data failed to predict influential organisms shaping community assembly. In sum, these methods defined the ecological roles of key species shaping community assembly and illuminated network design principles of microbial communities.

systems biology↗