Biology subjects

Lund, S. R.

Publications and source records attributed to Lund, S. R..

3 recordsLinked to original sources

bioRxiv2024

High-throughput Kinetics using Capillary Electrophoresis and Robotics (HiKER) platform used to Study T7, T3, and Sp6 RNA Polymerase Misincorporation

T7 RNA Polymerase (RNAP) is a well-studied and widely used enzyme with recent applications in the production of RNA vaccines. For over 50 years denaturing sequencing gels have been used as a key analysis tool for probing the kinetic mechanism of T7 RNAP nucleotide addition. However, sequencing gels are both slow and low throughput limiting their utility for comprehensive enzyme analysis. Here, we report the development of HiKER; (High-throughput Kinetics using Capillary Electrophoresis and Robotics) a high-throughput pipeline to quantitatively measure enzyme kinetics. We adapted a traditional polymerase misincorporation assay for fluorescent detection at scale allowing rapid estimates of RNAP misincorporation in different experimental conditions. In addition, high-throughput kinetics reactions were automated using an open-source OT-2 liquid handling robot. The platform allows multiple weeks worth of data to be collected in mere days. Using this platform, [~]1500 time points were collected in a single workday. T7 RNAP exhibited dramatic differences in both observed rate constant and amplitude depending on the mismatch examined. An average misincorporation frequency of [~]45 misincorporations per million bases was estimated using HiKER and is consistent with previous observations from next generation sequencing studies. Misincorporation time courses for T3 RNAP and Sp6 RNAP were similar to T7 RNAP suggesting conserved kinetic mechanisms. Interestingly, dramatic changes in the extent of misincorporation were observed in the three RNAPs depending on the mismatch. Extension from base mismatch experiments showed differences between T7, T3, and Sp6 RNAP. Sp6 RNAP was the slowest to extend from a mismatch followed by T7 RNAP and then T3 RNAP. Taken together the results presented here demonstrate the capabilities of HiKER to carry out high-throughput enzymology studies. Importantly, this pipeline and the corresponding analysis strategies are affordable, open-source, and broadly applicable to many enzymes.

biochemistry↗

bioRxiv2023

Virus-encoded glycosyltransferases hypermodify DNA with diverse glycans

Enzymatic modification of DNA nucleobases can coordinate gene expression, protection from nucleases, or mutagenesis. We recently discovered a new clade of phage-specific cytosine methyltransferase (MT) and 5-methylpyrimidine dioxygenase (5mYOX, e.g., TET) enzymes that produce 5-hydroxymethylcytosine (5hmC) as a precursor for additional post-replicative enzymatic hypermodifications on viral genomes. Here, we identify phage MT- and 5mYOX-dependent glycosyltransferase (GT) enzymes that catalyze linkage of diverse glycans directly onto 5hmC reactive nucleobase substrates. Using targeted bioinformatic mining of the phage metavirome databases, we discovered thousands of new biosynthetic gene clusters (BGCs) containing enzymes with predicted roles in cytosine sugar hypermodification. We developed a pathway reassembly platform for high-throughput functional screening of GT-containing BGCs, relying on the endogenous E. coli metabolome as a substrate pool. We successfully reconstituted a subset of phage BGCs and isolated novel and highly diverse sugar modifications appended to 5hmC, including mono-, di-, or tri-saccharide moieties comprised of hexose, N-acetylhexosamine or heptose sugars. Structural predictions and sugar product analyses suggest that phage GTs are related to host lipopolysaccharide, teichoic acid, and other small molecule biosynthesis enzymes and have been repurposed for DNA substrates. An expanded metagenomic search revealed hypermodification BGCs within gene neighborhoods containing phage structural proteins and putative genome defense systems. These findings enrich our knowledge of secondary modifications on DNA and the origins of corresponding sugar writer enzymes. Post-replicative cytosine hypermodification by virus-encoded GTs is discussed in the context of genome defense, DNA partitioning and virion assembly, and host-pathogen co-evolution.

microbiology↗

bioRxiv2023

Modification of DNA by a viral enzyme and charged tRNA

Bacteriophage enzymes synthesize varied and complex DNA hypermodifications. The enzyme encoded by the phage Mu gene mom is necessary for post-replicative carbamoylmethyl addition to the exocyclic amine of deoxyadenosine in DNA during the lytic phase of the viral life-cycle. The molecular details of this modification reaction, including the molecular origins of the modification itself, have long eluded understanding. Here, we demonstrate that Mom co-opts the translational machinery of the host by harvesting activated glycine from charged tRNAGly to hypermodify adenine. Based on this insight, we report the first in vitro reconstitution of the Mu hypermodification from purified components. Using isotope labeling, we demonstrate that the carbamoyl nitrogen of the Mom modification is derived from the N6 of adenine, indicating an on-base rearrangement of the N6 aminoacylation product, possibly via a cyclic intermediate. Informed by the X-ray crystal structure of Mom, we have probed the location of the active site, identified a novel insertion, and established substrate specificities of the Mom enzyme.

biochemistry↗