Yang, J. H. | bioRxiv Science

bioRxiv2022

A comprehensive update to the Mycobacterium tuberculosis H37Rv reference genome

H37Rv is the most widely used M. tuberculosis strain. Its genome is globally used as the M. tuberculosis reference sequence. We developed Bact-Builder, a pipeline that leverages consensus building to generate complete and highly accurate gap-closed bacterial genomes and applied it to three independently sequenced cultures of a parental H37Rv laboratory stock. Two of the 4,417,942 base-pair long H37Rv assemblies were 100% identical, with the third differing by a single nucleotide. Compared to the existing H37Rv reference, the new sequence contained approximately 6.4 kb additional base pairs encoding ten new regions. These regions included insertions in PE/PPE genes and new paralogs of esxN and esxJ, which were differentially expressed compared to the reference genes. Additional sequencing and assembly with Bact-Builder confirmed that all 10 regions were also present in widely accepted strains of H37Rv: NR123 and TMC102. Bact-builder shows promise as an improved method to perform extremely accurate and reproducible de novo assemblies of bacterial genomes. Furthermore, our findings provide important updates to the primary tuberculosis reference genome.

microbiology↗

bioRxiv2021

DNA double-strand break end synapsis by DNA loop extrusion

DNA double-strand breaks (DSBs) occur every cell cycle and must be efficiently repaired. Non-homologous end joining (NHEJ) is the dominant pathway for DSB repair in G1-phase. The first step of NHEJ is to bring the two DSB ends back into proximity (synapsis). However, although synapsis is generally assumed to occur through passive diffusion, we show here that passive diffusion is unlikely to be consistent with the speed and efficiency of NHEJ observed in cells. Instead, we hypothesize that DNA loop extrusion facilitates synapsis. By combining experimentally constrained simulations and theory, we show that the simplest loop extrusion model only modestly facilitates synapsis. Instead, a loop extrusion model with targeted loading of loop extruding factors (LEFs), a small portion of long-lived LEFs as well as LEF stabilization by boundary elements and DSB ends achieves fast synapsis with near 100% efficiency. We propose that loop extrusion plays an underappreciated role in DSB repair.

biophysics↗