Biology subjects

Spencer, J. J.

Publications and source records attributed to Spencer, J. J..

3 recordsLinked to original sources

bioRxiv2026

Hydrogen-bonding changes cause differences in imipenem breakdown activity in OXA-48 variants

The {beta}-lactamase OXA-48 efficiently hydrolyses carbapenem antibiotics, especially imipenem. Carbapenem resistance is a rising clinical concern, and is frequently associated with OXA-48 and its variants. OXA-48 variants carrying different mutations in the {beta}5-{beta}6 loop differ in hydrolytic activity towards imipenem. OXA-517 has a higher KM, but similar kcat for imipenem hydrolysis, compared to OXA-48, whereas OXA-163 and -405, which have similar mutations in the {beta}5-{beta}6 loop, are less active. Multiscale simulations (using quantum mechanics/molecular mechanics, QM/MM) of deacylation of the respective imipenem acylenzymes show this to be most efficient when the deacylating water (DW) acts as a hydrogen bond (H-bond) donor to imipenem, and the carboxylated Lys73 base is less hydrated. Calculated barriers for deacylation correlate very well with experimental data, but for OXA-163 and -405 only when DW acts as a H-bond acceptor. Dynamics simulations of imipenem acylenzyme complexes show that mutations in the {beta}5-{beta}6 loop change the active site H-bond network. In OXA-48, the DW H-bonding pattern linked to high activity is more frequently sampled, and in OXA-517 it is stabilised through H-bonding to Thr213; explaining the higher kcat values compared to OXA-163 and -405, where this is not the case. Furthermore, simulations of non-covalent imipenem complexes indicate that increased KM for OXA-517 is linked to lower binding affinity, caused by repositioning of bound imipenem. Our work identifies the molecular basis for differences in imipenem hydrolytic activity between OXA-48 variants, offering detailed insights into how active site interactions alter the dynamics and reaction efficiencies related to antibiotic resistance.

biochemistry↗

bioRxiv2024

Structural basis of gap-filling DNA synthesis in the nucleosome by DNA Polymerase β

Single-strand breaks (SSBs) are one of the most prevalent forms of DNA damage found in the chromatinized genome and are repaired by direct single-strand break repair (SSBR) or base excision repair (BER). DNA polymerase beta (Pol {beta}) is the primary enzyme responsible for processing the 1-nt gap intermediate in chromatin during SSBR and BER. However, the mechanism used by Pol {beta} to process a 1-nt gap in the context of the nucleosome and chromatin remains poorly understood. Here, we use biochemical assays and cryogenic electron microscopy (cryo-EM) to determine the kinetic and structural basis of gap-filling DNA synthesis in the nucleosome by Pol {beta}. Kinetic analysis identified that gap-filling DNA synthesis in the nucleosome by Pol {beta} is position-dependent, where solvent exposed 1-nt gaps are processed more efficiently than histone-occluded 1-nt gaps. A series of cryo-EM structures of Pol {beta} bound to a solvent-exposed 1-nt gap in the nucleosome reveal a global DNA sculpting mechanism for 1-nt gap recognition, which is mediated by sequential engagement of the Pol {beta} lyase domain and polymerase domain. Finally, cryo-EM structures of Pol {beta} bound to 1-nt gaps at two additional positions in the nucleosomal DNA define the structural basis for position-dependent nucleotide insertion in the nucleosome. This work establishes the mechanism used by Pol {beta} for processing 1-nt gaps in the nucleosome during SSBR and BER, providing fundamental insight into DNA repair in chromatin.

biochemistry↗

bioRxiv2022

Structural basis for APE1 processing DNA damage in the nucleosome

Genomic DNA is continually exposed to endogenous and exogenous factors that promote DNA damage. Eukaryotic genomic DNA is packaged into nucleosomes, which present a barrier to accessing and effectively repairing DNA damage. The mechanisms by which DNA repair proteins overcome this barrier to repair DNA damage in the nucleosome and protect genomic stability is unknown. Here, we determine how the base excision repair (BER) endonuclease AP-endonuclease 1 (APE1) recognizes and cleaves DNA damage in the nucleosome. Kinetic assays determined that APE1 cleaves solvent-exposed AP sites in the nucleosome with 3 - 6 orders of magnitude higher efficiency than occluded AP sites. A cryo-electron microscopy structure of APE1 bound to a nucleosome containing a solvent-exposed AP site identified that APE1 uses a DNA sculpting mechanism for AP site recognition, where APE1 bends the nucleosomal DNA to access the AP site. Notably, additional biochemical and structural characterization of occluded AP sites identified contacts between the nucleosomal DNA and histone octamer that prevent efficient processing of the AP site by APE1. These findings provide a rationale for the position-dependent activity of BER proteins in the nucleosome and suggests the ability of BER proteins to sculpt nucleosomal DNA drives efficient BER in chromatin.

biochemistry↗