Mitsunobu, H. | bioRxiv Science

bioRxiv2024

Replication of DNA containing trinucleotide repeats by the bacteriophage T7 replisome

Trinucleotide repeats in the human genome are implicated in various neurodegenerative diseases. The tendency of these repetitive DNA sequences to form non-B DNA structures can cause abnormal replication, leading to genomic instability. This instability contributes to disease progression, though the underlying mechanisms are not fully understood. We investigated the replication of DNA containing CAG and CTG trinucleotide repeats using individual components of the T7 bacteriophage replication machinery, as well as the complete replisome. Our results show that repeats in linear single-stranded DNA (ssDNA) inhibit the activity of T7 DNA polymerase and ssDNA-binding proteins, with a more pronounced effect observed in CTG repeats compared to CAG repeats. Minicircle templates containing CTG repeats exhibited robust DNA synthesis on both the leading and lagging strands, though synthesis was not enhanced by the T7 gene 2.5 ssDNA-binding protein. The lagging strand products generated from the CTG repeat minicircle were significantly longer than those from random sequence templates, and their lengths were not extended by the presence of T7 gene 2.5 protein. When the repeated sequences were incorporated into the T7 phage genome, heterogeneity was observed downstream of the repeats, depending on their length. We propose that aberrant extension occurs predominantly in the lagging strand, driven by dynamic interactions between the repeated sequences and the DNA replisome. This study may provide a foundation for understanding the mechanisms underlying the extension or deletion of repetitive genomic regions.

biochemistry↗

bioRxiv2024

Development of a highly efficient base editing system for Lactobacilli to improve probiotics and dissect essential functions

Lactobacilli play essential roles in the food industry and are increasingly explored for their potential as probiotics and therapeutic agents. Beneficial strains are primarily isolated from various natural sources including healthy human bodies, and undergo rigorous characterization and safety evaluations. Genomic and genetic information has increasingly accumulated and been linked to their various functions, to which transgenic approaches are being performed to verify crucial genes. In order to reasonably develop more useful strains, beneficial traits need to be introduced into any given strains and enhanced or combined. However, for practical use as probiotics or foods, organisms with transgene are hardly acceptable. Here, we have introduced the base editing Target-AID system specifically for Lactobacilli, enabling precise installation of point mutations without donor DNA and at multiple genomic loci simultaneously. Lactiplantibacillus plantarum has been successfully engineered to reduce production of imidazole propionate, which has been reported to be associated with type 2 diabetes. Additionally, this system enabled transient knock-out of an essential gene, such as one involved in cell division showing severe filamentous cell phenotype, providing a unique approach for dissecting essential gene function. ImportanceThis work provides highly efficient and multiplexable base editing system that installs precise point mutations in the genomes of the two major Lactobacilli strains. As the advanced CRISPR technology so-called non-cleaving genome editing, base editing is less toxic and does not integrate any foreign DNA into the genomes. Our approaches pave the way for dissecting and improving probiotics and food-grade microbes, ultimately creating better human health.

microbiology↗