Biology subjects

van Laar, T.

Publications and source records attributed to van Laar, T..

6 recordsLinked to original sources

bioRxiv2026

Systematic engineering and machine learning analysis of intrinsic terminators reveal crucial nucleotides directly upstream of the terminator hairpin.

Transcriptional termination efficiency is considered an important parameter for finetuning bacterial gene expression. Still, the design principles that determine transcription termination efficiency remain poorly understood. In this study, we aimed to investigate the impact of the 3 untranslated region (3UTR) on gene expression in Escherichia coli and other bacteria. First, 3UTR variant sequences were generated, with randomized 30 bp sequences inserted between the STOP-codon and an intrinsic terminator, consisting of a GC-rich hairpin and a downstream poly(U)-tail. Using three reporter genes, it was found that different 3UTR sequences resulted in an up to five-fold difference in protein production, independent of the upstream coding sequence. The highest protein production was achieved when an adenosine was present directly upstream of the terminator hairpin. This was consolidated by systematic substitution of key nucleotides of the terminator and assessing their effect on mRNA and protein levels. Subsequently, we developed a predictive random forest machine learning model trained on the termination efficiency of different natural and synthetic terminator sequences, revealing an important role for the nucleotides directly upstream of the terminator hairpin. Altogether, this study showed that an additional adenosine nucleotide upstream of the terminator hairpin leads to improved protein production while reducing terminator read-through. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/736697v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@d8a976org.highwire.dtl.DTLVardef@5d8269org.highwire.dtl.DTLVardef@11cc3e3org.highwire.dtl.DTLVardef@180a305_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

A human multilineage gut organoid model for Parkinson disease

Emerging evidence links gut dysfunction to Parkinson disease (PD) pathogenesis, yet human models to study gut-related mechanisms are lacking. We developed a human intestinal organoid model incorporating PD-relevant cell types. Using control and PD patient-derived pluripotent stem cells, we generated intestinal epithelial organoids and vagal neural crest cells, then co-cultured them into assembloids. The resultant structures featured lumen-forming polarized epithelial monolayers with enteroendocrine cells, contractile subepithelial myofibroblast-like layers, and neuroglial networks containing dopaminergic and cholinergic enteric neurons. Notably, assembloids from a PD patient carrying the GBA1-E326K variant exhibited progressive -synuclein accumulation in non-enteroendocrine epithelial cells. This model recapitulates key gut architecture and PD-associated phenotypes, offering a physiologically relevant platform for mechanistic studies and therapeutic discovery targeting gut-brain pathways in PD.

cell biology↗

bioRxiv2023

A chromatinized origin reduces ORC mobility and spatially constrains MCM

Chromatin replication involves the assembly and activity of the replisome within the nucleosomal landscape. At the core of the replisome is the Mcm2-7 complex (MCM), which is loaded onto DNA after binding to the Origin Recognition Complex (ORC). In yeast, ORC is a dynamic protein that diffuses rapidly along DNA, unless halted by origin recognition sequences. However, less is known about the dynamics of ORC proteins in the presence of nucleosomes and attendant consequences for MCM loading. To address this, we harnessed an in vitro single-molecule approach to interrogate a chromatinized origin of replication. We find that ORC binds the origin of replication with similar efficiency independently of whether the origin is chromatinized, despite ORC mobility being reduced by the presence of nucleosomes. Recruitment of MCM also proceeds efficiently on a chromatinized origin, but subsequent movement of MCM away from the origin is severely constrained. These findings suggest that chromatinized origins in yeast are essential for the local retention of MCM, which may facilitate subsequent assembly of the replisome.

biophysics↗

bioRxiv2022

Nucleotide binding halts diffusion of the eukaryotic replicative helicase during activation

The eukaryotic replicative helicase CMG centrally orchestrates the replisome and leads the way at the front of replication forks1. Understanding the motion of CMG on the DNA is therefore key to our understanding of DNA replication. In vivo, CMG is assembled and activated through a cell-cycle-regulated mechanism involving 36 polypeptides that has been reconstituted from purified proteins in ensemble biochemical studies2,3. Conversely, single-molecule studies of CMG motion have thus far4-6 relied on pre-formed CMG assembled through an unknown mechanism upon overexpression of individual constituents7,8. Here, we report the first activation at the single-molecule level of CMG fully reconstituted from purified yeast proteins and the quantification of its motion. We observe that CMG can move on DNA in two ways: by unidirectional translocation and by diffusion. We demonstrate that CMG preferentially exhibits unidirectional translocation in the presence of ATP, whereas it preferentially exhibits diffusive motion in the absence of ATP. We also demonstrate that nucleotide binding halts diffusive CMG. Taken together, our findings support a mechanism by which nucleotide binding allows newly assembled CMG to engage with the DNA within its central channel without melting it, halting its diffusion and facilitating the initial DNA melting required to initiate DNA replication.

biophysics↗

bioRxiv2022

CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 cooperates with the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitution, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Pol{varepsilon}) for PCNA binding, but not with the lagging strand DNA polymerase Delta (Pol{delta}). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand both processes are coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication.

biochemistry↗

bioRxiv2021

SMC complexes can traverse physical roadblocks bigger than their ring size

The ring-shaped structural-maintenance-of-chromosomes (SMC) complexes condensin and cohesin extrude loops of DNA as a key motif in chromosome organization. It remains, how ever, unclear how these SMC motor proteins can extrude DNA loops in chromatin that is bound with proteins. Here, using in vitro single-molecule visualization, we show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to DNA loop extrusion by yeast condensin. Strikingly, we find that even DNA-bound nanoparticles as large as 200 nm, much bigger than the SMC ring size, can be translocated into DNA loops during condensin-driven extrusion. Similarly, human cohesin can pass 200 nm particles during loop extrusion, which even occurs for a single-chain version of cohesin in which the ring-forming subunits are covalently linked and cannot open up to entrap DNA. These findings disqualify all common loop-extrusion models where DNA passes through the SMC rings (pseudo)topologically, and instead point to a nontopological mechanism for DNA loop extrusion. One-sentence summaryHuge DNA-bound roadblocks can be incorporated into SMC-extruded DNA loops, pointing to a nontopological mechanism for loop extrusion.

biophysics↗