Biology subjects

Phipps, J.

Publications and source records attributed to Phipps, J..

4 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↗

bioRxiv2023

Cohesin complex oligomerization maintains end-tethering at DNA double-strand breaks

DNA double-strand breaks (DSB) must be repaired to ensure genome stability. Crucially, DSB ends must be kept together for timely repair. In Saccharomyces cerevisiae, two poorly understood pathways mediate DSB end-tethering. One employs the Mre11-Rad50-Xrs2 (MRX) complex to physically bridge DSB ends. Another requires the conversion of DSB ends into single-strand DNA (ssDNA) by Exo1, but the bridging proteins are unknown. We uncover that cohesin, its loader and Smc5/6 act with Exo1 to tether DSB ends. Remarkably, cohesin specifically impaired in oligomerization fails to tether DSB ends, revealing a new function for cohesin oligomerization. In addition to the known importance of sister chromatid cohesion, microscopy-based microfluidic experiments unveil a new role for cohesin in repair by ensuring DSB end-tethering. Altogether, our findings demonstrate that oligomerization of cohesin prevents DSB end separation and promotes DSB repair, revealing a novel mode of action and role for cohesin in safeguarding genome integrity.

molecular biology↗

bioRxiv2023

Telomere protein arrays stall DNA loop extrusion by condensin

DNA loop extrusion by SMC proteins is a key process underlying chromosomal organization. It is unknown how loop extruders interact with telomeres where chromosome ends are covered with a dense array of tens of neighboring DNA-binding proteins. Using complementary in vivo and in vitro single-molecule approaches, we study the interaction between loop-extruding condensin and arrays of Rap1, the double-stranded-DNA-binding telomeric protein of Saccharomyces cerevisiae. We show that dense linear Rap1 arrays can completely halt DNA loop extrusion, where the blocking efficiency depends on the array length and the DNA gap size between neighboring proteins. In cells, Rap1 arrays in the chromosome are found to act as contact insulators and to accumulate condensin at their borders, with direct implications for the resolution of dicentric chromosomes produced by telomere fusions. Our findings show that linear arrays of DNA-bound proteins can efficiently halt DNA loop extrusion by SMC proteins, which may impact a wide range of cellular processes from telomere functions to transcription and DNA repair.

molecular biology↗