Biology subjects

Sayers, J. R.

Publications and source records attributed to Sayers, J. R..

3 recordsLinked to original sources

bioRxiv2024

Flap endonuclease Substrate Interactions studied using Dynamic Single-Molecule Atomic Force Microscopy

Flap endonucleases (FENs) recognise and cleave DNA substrates containing a 5-single-strand (ss) of nucleic acid branching off a double-stranded (ds) DNA to yield a nicked duplex during DNA replication. Dynamic Atomic Force Microscopy of an inactive FEN mutant complexed with branched DNA revealed mobilisation of immobilised DNA, indicating that protein interaction affected substrate conformation and disrupted the forces that anchored it to the poly-L-ornithine -treated mica surface. Enzymatically-active FEN was seen intermittently binding DNA, altering its conformation and cleaving the ssDNA branch. We developed a method using motion tracking for quantifying the movement of DNA sections, by visually segmenting DNA and tracking each segment to recognise the DNA sections most affected by the protein. It was found that whilst bound, FEN caused localised DNA bending, and changes in DNA shape were witnessed in the short time span of the proteins appearance close to the nucleic acid, followed by protein adsorption on the mica surface. The results provide the first dynamic observations of FEN-DNA interaction. FEN initially binds to the dsDNA, slides to find the ds/ssDNA junction, and the 5 ssDNA likely threads through a hole in the enzyme which leads to enzymatic hydrolysis of the branched substrate.

biochemistry↗

bioRxiv2024

Efficient overexpression and purification of SARS-CoV-2 Nucleocapsid proteins in Escherichia coli

The fundamental biology of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap in Escherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducible expression vector carrying a regulated bacteriophage T5 synthetic promoter controlled by lac operator binding sites. The constructs were used to express Ncap proteins and protocols developed which allow efficient production of purified Ncap with yields of over 200 mg per litre of culture media. These proteins were deployed in ELISA assays to allow comparison of their responses to human sera. Our results suggest that there was no detectable difference between the 6His-tagged and untagged original Ncap proteins but there may be a slight loss of sensitivity of sera to other Ncap isolates.

biochemistry↗

bioRxiv2022

Centriole growth is not limited by a finite pool of components, but is limited by the Cdk1/Cyclin-dependent phosphorylation of Ana2/STIL

Centrioles duplicate once per cell cycle but it is unclear how daughter centrioles assemble at the right time and place and grow to the right size. Here we show that in early Drosophila embryos the cytoplasmic concentrations of the key centriole assembly proteins Asl, Plk4, Ana2, Sas-6 and Sas-4 are low, but remain constant throughout the assembly process-- indicating that none of them are limiting for centriole assembly. The cytoplasmic diffusion rate of Ana2/STIL, however, increased significantly towards the end of S-phase as Cdk/Cyclin activity in the embryo increased. A mutant form of Ana2 that cannot be phosphorylated by Cdk/Cyclins did not exhibit the diffusion rate change, and allowed daughter centrioles to grow for an extended period. Thus, the Cdk/Cyclin-dependent phosphorylation of cytoplasmic Ana2 seems to reduce the efficiency of daughter centriole assembly towards the end of S-phase. This helps to ensure that daughter centrioles stop growing at the correct time, and presumably also helps to explain why centrioles cannot duplicate during mitosis when Cdk/Cyclin activity is high.

cell biology↗