Biology subjects

Solano, B.

Publications and source records attributed to Solano, B..

3 recordsLinked to original sources

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↗

bioRxiv2020

Induced copy-back RNA synthesis as a novel therapeutic mechanism against RNA viruses

The viral RNA-dependent RNA polymerase (RdRp) is a well-established target for development of broad-spectrum antiviral therapeutics. Incorporation of ribonucleotide analogues by the RdRp will either cause termination of RNA synthesis or mutagenesis of the RNA product. We demonstrated recently that incorporation of a pyrazine-carboxamide ribonucleotide into nascent RNA leads to pausing and backtracking of the elongating RdRp. Here, we provide evidence for the single-stranded RNA product of backtracking serving as an intermediate in RdRp-catalyzed, template-switching reactions. This intermediate is used for both intramolecular template-switching (copy-back RNA synthesis) and intermolecular template-switching (homologous RNA recombination). The use of a magnetic-tweezers platform to monitor RdRp elongation dynamics permitted direct observation of copy-back synthesis and illuminated properties of the RdRp that promote copy-back synthesis, including stability of the RdRp-nascent-RNA complex and the dimensions of the RdRp nucleic-acid-binding channel. In cells, recombination was stimulated by the presence of a pyrazine-carboxamide ribonucleotide. The effect of the drug on recombination was diminished for a recombination-defective virus, but this virus was not resistant to the drug. The discovery that a ribonucleotide analogue can induce copy-back RNA synthesis suggests that this third mechanistic class of compounds may function by promoting formation of defective viral genomes. This study identifies RdRp-catalyzed intra- and intermolecular template switching as a viable new mechanistic target with potentially broad-spectrum appeal.

microbiology↗