Biology subjects

Bloom, L.

Publications and source records attributed to Bloom, L..

5 recordsLinked to original sources

bioRxiv2025

A Novel {psi}-χ Fusion Protein for Unravelling the Contributions of χ to DNA Replication and Repair

Faithful DNA replication in Escherichia coli requires the DNA polymerase III holoenzyme (DNA pol III HE) and its clamp loader complex, which couples processive DNA synthesis with {beta}-clamp loading. The clamp loader accessory subunits {chi} and {psi} link single-stranded DNA-binding protein (SSB) to the replisome, stabilizing replication on SSB-coated templates through interactions with the SSB C-terminal tail. Chi has also been implicated in tolerance to the chain-terminating nucleoside analog azidothymidine (AZT), though whether this function depends on {chi} within DNA pol III HE or on its independent interaction with the YoaA helicase remains unclear. To address this, we engineered {psi}-{chi} fusion proteins with flexible glycine-serine linkers to tether the two subunits while preserving folding and activity. Both fusions were biochemically competent, supporting ATP hydrolysis and clamp loading on SSB-coated DNA. In vivo, however, neither the {psi}-GS12-{chi} fusion nor expression of a {psi}{chi} operon restored AZT tolerance in {Delta}holC cells, whereas expression of {chi} alone was sufficient. Fusion expression impaired growth in both wild-type (WT) and {Delta}holC backgrounds, a phenotype alleviated by disrupting {chi}-SSB binding. These findings support a model in which {chi} must dynamically engage SSB and YoaA outside of the clamp loader to promote AZT tolerance, highlighting the importance of regulated {chi}-SSB interactions in genome maintenance.

biochemistry↗

bioRxiv2024

Single-stranded DNA binding protein hitches a ride with the Escherichia coli YoaA-χ helicase

The Escherichia coli XPD/Rad3-like helicase, YoaA, and DNA polymerase III subunit, {chi}, are involved in E. coli DNA damage tolerance and repair. YoaA and {chi} promote tolerance to the DNA chain-terminator, 3{square}-azidothymidine (AZT), and together form the functional helicase complex, YoaA-{chi}. How YoaA-{chi} contributes to DNA damage tolerance is not well understood. E. coli single-stranded DNA binding protein (SSB) accumulates at stalled replication forks, and the SSB-{chi} interaction is required to promote AZT tolerance via an unknown mechanism. YoaA-{chi} and SSB interactions were investigated in vitro to better understand this DNA damage tolerance mechanism, and we discovered YoaA-{chi} and SSB have a functional interaction. SSB confers a substrate-specific effect on the helicase activity of YoaA-{chi}, barely affecting YoaA-{chi} on an overhang DNA substrate but inhibiting YoaA-{chi} on forked DNA. A paralog helicase, DinG, unwinds SSB-bound DNA in a similar manner to YoaA-{chi} on the substrates tested. Through use of ensemble experiments, we believe SSB binds behind YoaA-{chi} relative to the DNA ds/ss junction and show via single-molecule assays that SSB translocates along ssDNA with YoaA-{chi}. This is, to our knowledge, the first demonstration of a mechanoenzyme pulling SSB along ssDNA.

biochemistry↗

bioRxiv2023

Increased FAN1 expression by mRNA-LNP attenuates CAG repeat expansion in Huntington patients' iPSC-derived astrocytes

Expansion of repeat sequences within the human genome can lead to disease pathogenesis, such as Huntingtons Disease, primarily affecting the nervous system. Genome-wide association studies (GWAS) of age-at-onset in Huntingtons disease (HD) patients demonstrated DNA mismatch repair (MMR) genes are modifiers of somatic expansion and may be potential therapeutic targets for repeat expansion (RE) disorders. FAN1, a Fanconi anemia-associated nuclease, has been reported as an influencer of repeat expansion in the RE mouse models. Here, we show the first demonstration that FAN1 knock-out in HD patient-derived fibroblasts and results in increased CAG repeat length. We also develop a robust novel cell-based platform using stem cell technology to produce the HD patients iPSC-derived astrocytes (iAstro). This platform is a disease-relevant system and has a significantly wider assay window, making it more suitable to assess the effect of gene modulation on CAG repeats. A substantial and exponential increase in repeat instability was exhibited in this HD patients iPSC-derived astrocytes platform. Over-expression of FAN1 protein via FAN1 plasmid transfection in this platform reduced CAG repeat instability, suggesting that upregulation of FAN1 protein may have a potential protective effect in CAG repeat expansion for a therapeutic setting. We leveraged the mRNA-LNP modality to enhance FAN1 protein expression and revealed that codon-optimized FAN1 mRNA-LNP robustly prevented increased CAG repeat in HD patients iPSC-derived astrocytes platform. The data from these cell-based platforms highlight that FAN1 plays a protective role in attenuating expanded somatic HTT CAG repeats and shed light on new therapeutic directions against repeat expansion disorders.

neuroscience↗

bioRxiv2020

Potassium Glutamate and Glycine Betaine Induce Self-Assembly of Sliding Clamps into Higher Order Oligomers

Sliding clamps are oligomeric ring-shaped proteins that increase the efficiency of DNA replication. The stability of the Escherichia coli {beta}-clamp, a homodimer, is particularly remarkable. The dissociation equilibrium constant of {beta} is of the order of 10 pM in buffers of moderate ionic strength. Coulombic electrostatic interactions have been shown to contribute to this remarkable stability. Increasing NaCl concentration in the assay buffer results in decreased dimer stability and faster subunit dissociation kinetics in a way consistent with simple charge-screening models. Here, we examine non-Coulombic ionic effects on the oligomerization properties of sliding clamps. Replacing NaCl by KGlu, the primary cytoplasmic salt in E. coli, results in the formation of assemblies that involve two or more rings stacked face-to-face. Results can be quantitatively explained on the basis of unfavorable interactions between KGlu and the functional groups on the protein surface, which drive biomolecular processes that bury exposed surface. Similar results were obtained with the S. cerevisiae PCNA sliding clamp, suggesting that KGlu effects are not specific to {beta}. Clamp association is also promoted by glycine betaine, a zwitterionic compound that accumulates intracellularly when E. coli is exposed to high concentrations of extracellular solute. Possible biological implications are discussed.

biophysics↗

bioRxiv2018

Novel organelle anion channels formed by chromogranin B drive normal granule maturation in endocrine cells

All endocrine cells need an anion conductance for maturation of secretory granules. Identity of this family of anion channels has been elusive for forty years. We now show that a family of granule protein, CHGB, serves the long-sought conductance. CHGB interacts with membranes through two amphipathic helices, and forms a chloride channel with large conductance and high anion selectivity. Fast kinetics and high cooperativity suggest that CHGB tetramerizes to form a functional channel. Nonfunctional mutants separate CHGBs function in granule maturation from that in granule biogenesis. In neuroendocrine cells, CHGB channel and a H+-ATPase drives normal insulin maturation inside or dopamine loading into secretory granules. CHGBs tight membrane-association after exocytotic release of secretory granules separates its intracellular function from extracellular functions of its proteolytic peptides. CHGB-null mice show consistent impairment of granule acidification in pancreatic beta-cells. These findings together support that the phylogenetically conserved CHGB proteins constitute a new family of organelle chloride channels in the regulated secretory pathway among various endocrine cells.

cell biology↗