Dubiel, K. | bioRxiv Science

bioRxiv2023

Spatiotemporal dynamics of single-stranded DNA intermediates in Escherichia coli

Single-stranded DNA gaps form within the E. coli chromosome during replication, repair and recombination. However, information about the extent of ssDNA creation in the genome is limited. To complement a recent whole-genome sequencing study revealing ssDNA gap genomic distribution, size, and frequency, we used fluorescence microscopy to monitor the spatiotemporal dynamics of single-stranded DNA within live E. coli cells. The ssDNA was marked by a functional fluorescent protein fusion of the SSB protein that replaces the wild type SSB. During log-phase growth the SSB fusion produces a mixture of punctate foci and diffuse fluorescence spread throughout the cytosol. Many foci are clustered. Fluorescent markers of DNA polymerase III frequently co-localize with SSB foci, often localizing to the outer edge of the large SSB features. Novel SSB-enriched features form and resolve regularly during normal growth. UV irradiation induces a rapid increase in SSB foci intensity and produces large features composed of multiple partially overlapping foci. The results provide a critical baseline for further exploration of ssDNA generation during DNA metabolism. Alterations in the patterns seen in a mutant lacking RecB function tentatively suggest associations of particular SSB features with the repair of double strand breaks and post-replication gaps.

biophysics↗

bioRxiv2019

Evolution of (p)ppGpp-HPRT regulation through diversification of an allosteric oligomeric interaction

The signaling ligand (p)ppGpp binds diverse targets across bacteria, yet the mechanistic and evolutionary basis underlying these ligand-protein interactions remains poorly understood. Here we identify a novel (p)ppGpp binding motif in the enzyme HPRT, where (p)ppGpp shares identical binding residues for PRPP and nucleobase substrates to regulate purine homeostasis. Intriguingly, HPRTs across species share the conserved binding site yet strongly differ in ligand binding, from strong inhibition by basal (p)ppGpp levels to weak regulation at induced concentrations. Surprisingly, strong ligand binding requires an HPRT dimer-dimer interaction that allosterically opens the (p)ppGpp pocket. This dimer-dimer interaction is absent in the common ancestor but evolved to favor (p)ppGpp binding in the vast majority of bacteria. We propose that the evolutionary plasticity of oligomeric interfaces enables allosteric adjustment of ligand regulation, bypassing constraints of the ligand binding site. Since most ligands bind near protein-protein interfaces, this principle likely extends to other protein-ligand interactions.

biochemistry↗