Biology subjects

Tillett, Z.

Publications and source records attributed to Tillett, Z..

2 recordsLinked to original sources

bioRxiv2026

DNA Ligases Discriminate Between Natural and Non-Natural Base Pairs

Artificially Expanded Genetic Information Systems (AEGIS) increase the information content of nucleic acids by including new nucleobase pairings that are orthogonal to those of canonical Watson-Crick nucleobases. DNA ligases do not form direct interactions with the nucleobases during catalytic turnover, suggesting that these enzymes should efficiently and faithfully join double-stranded AEGIS substrates. Here we report the systematic investigation into the validity of this hypothesis for structurally-diverse DNA ligases employing substrates built from the eight nucleotide hachimoji genetic alphabet, where orthogonality is achieved by rearranging the hydrogen bonding patterns seen in canonical Watson-Crick pairs. We find that single, or multiple, non-canonical bases are well tolerated at the 5-end of the nick. However, tracts of consecutive non-canonical bases at the 3-end of the break significantly decrease ligation efficiency or abolish it altogether. Possible reasons for this apparent bias against non-canonical nucleobases could include incompatibility in electrostatic interactions between the ligase active site and the non-canonical substrates or altered conformational preferences and/or dynamics in key catalytic intermediates. We also observe single hachimoji mismatches are ligated more frequently than mis paired canonical bases, potentially due to promiscuous pairing of tautomeric forms of the non-canonical bases.

biochemistry↗

bioRxiv2025

Mechanism of NanR transcriptional activation of sialic acid metabolism in Streptococcus pneumoniae.

In Streptococcus pneumoniae, the RpiR transcriptional regulator NanR (SpNanR) senses sialic acid in the environment and upregulates transcription of the nan and siaA operons to increase uptake and metabolism of sialic acid. The molecular basis of this activation is unknown. Here, we demonstrate that SpNanR binds N-acetylmannosamine-6-phosphate, a metabolite of sialic acid catabolism. SpNanR exists in a dimer-tetramer equilibrium, and N-acetylmannosamine-6-phosphate binding strongly stabilizes the tetramer. Crystal structures and site-specific substitutions demonstrate that N-acetylmannosamine-6-phosphate bridges and stabilizes the SpNanR tetramer. SpNanR binds its DNA recognition sequence with nanomolar affinity. Notably, the effector N-acetylmannosamine-6-phosphate does not affect the affinity of SpNanR for DNA. The DNA binding domains are not structurally coupled to the sugar isomerase domains, explaining why N-acetylmannosamine-6-phosphate binding does not affect DNA binding. Structural analysis reveals that sequence specificity arises through distortion of B-DNA and an unusual {pi}-stack formed by two arginine residues in the minor groove, while affinity is driven by backbone contacts. We propose a mechanism by which S. pneumoniae regulates sialic acid metabolism, consistent with our biophysical experiments and in vivo regulatory behavior. These findings define a unique activation mechanism for an RpiR regulator and provide new insights into carbohydrate-responsive gene regulation in pneumococci.

molecular biology↗