Biology subjects

Phelps, S.

Publications and source records attributed to Phelps, S..

6 recordsLinked to original sources

bioRxiv2026

Mismatch repair MLH complexes make distinct contributions to post-replicative mismatch repair versus trinucleotide repeat expansions

Mismatch repair (MMR) is a highly conserved DNA repair pathway that promotes genome stability by directing the repair of errors in DNA replication. In Saccharomyces cerevisiae, MMR is initiated by either Msh2-Msh3 or Msh2-Msh6, via recognition of insertion deletion loops (IDLs; up to [~] 17 nucleotides) and misincorporation events, respectively. Both complexes recognize and bind small (1-2 nucleotide) IDLs. Once bound, MSH complexes recruit one or more downstream MLH complexes to continue repair: Mlh1-Pms1, Mlh1-Mlh2 and/or Mlh1-Mlh3. Msh2-Msh3 also promotes CAG trinucleotide repeat (TNR) expansions through specific DNA-binding to TNR DNA structures, followed by recruitment of MLH complexes. These expansions lead to genome instability that causes neurodegenerative diseases such as Huntingtons Disease in humans. Here, we defined a hierarchy of MLH function in these Msh2-Msh3-mediated pathways in vivo in S. cerevisiae. We determined that Mlh1-Pms1 is the primary MLH complex required in Msh2-Msh3-mediated MMR. In contrast, all three MLH complexes were required to promote CAG expansions, with loss of Mlh1-Pms1 or Mlh1-Mlh2 exhibiting the strongest effects. Mutations in PMS1 and MLH3 were synergistic. We propose a model in which Mlh1-Pms1 is primarily responsible for "appropriate" Msh2-Msh3-mediated MMR, while all three MLH complexes collaborate specifically in the presence of CAG structure, to promote a "pathogenic" Msh2-Msh3-mediated pathway that leads to expansions. Our model highlights the importance of DNA structure-dependent conformations in modulating MLH function.

genetics↗

bioRxiv2025

NitrOFF: An engineered fluorescent biosensor to illuminate nitrate transport in living cells

The duality of nitrate is nowhere best exemplified than in human physiology - a detrimental pollutant but also a protective nutrient and signaling ion - particularly as connected to reactive nitrogen oxides. Aside from limited insights into nitrate uptake and storage, foundational nitrate biology has lagged. Genetically encoded fluorescent biosensors can address this gap with real-time imaging. However, imaging technologies for mammalian cell applications remain rare. Here, we set out to design and engineer a two-domain chimera fusing the split green fluorescent protein EGFP and the nitrate recognition domain NreA from Staphylococcus carnosus. Over 7 rounds of directed evolution, 15 mutations were accumulated resulting in the functional biosensor NitrOFF. NitrOFF has a high degree of allosteric communication between the domains reflected in a turn-off intensiometric response (Kd {approx} 9 {micro}M). This was further reinforced by X-ray crystal structures of apo and nitrate bound NitrOFF, which revealed that the two domains undergo a large-scale conformational rearrangement that changes the relative positioning of the EGFP and NreA domains by 68.4{degrees}. Such a dramatic difference was triggered by the formation of a long helix at the engineered linker connecting the two domains, peeling the {beta}7 strand off the EGFP and thus extinguishing the fluorescence upon nitrate binding. Finally, as a proof-of-concept, we highlighted the utility of this first-generation biosensor to monitor exogenous nitrate uptake and modulation in a human embryonic kidney (HEK) 293 cell line. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/644677v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1ba853eorg.highwire.dtl.DTLVardef@81bed9org.highwire.dtl.DTLVardef@18a313org.highwire.dtl.DTLVardef@17a5e80_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Directed Evolution of a Genetically Encoded Indicator for Chloride

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/624492v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@8a9309org.highwire.dtl.DTLVardef@1bb6770org.highwire.dtl.DTLVardef@840dd4org.highwire.dtl.DTLVardef@1a7e2cb_HPS_FORMAT_FIGEXP M_FIG C_FIG Inarguably, the green fluorescent protein (GFP) family is an exemplary model for protein engineering, accessing a range of unparalleled functions and utility in biology. The first variant to recognize and provide an optical output of chloride in living cells was serendipitously uncovered more than 25 years ago. Since then, researchers have actively expanded the potential of GFP indicators for chloride through site-directed and combinatorial site-saturation mutagenesis, along with chimeragenesis. However, to date, the power of directed evolution has yet to be unleashed. As a proof-of-concept, here, we use random mutagenesis paired with anion walking to engineer a chloride-insensitive fluorescent protein named OFPxm into a functional indicator named ChlorOFF. The sampled mutational landscape unveils an evolutionary convergent solution at one position in the anion binding pocket and nine other mutations across eight positions, of which only one has been previously linked to chloride sensing potential in the GFP family.

biochemistry↗

bioRxiv2024

Androgen receptor modulation of vocal circuitry in Alston's singing mouse

Animal courtship and aggressive displays are dramatic, often sexually dimorphic behaviors that require the coordinated modulation of diverse motivational and motor circuits. In Alstons singing mice, a novel and elaborate advertisement vocalization is sexually dimorphic and steroid sensitive (Scotinomys teguina). Males sing more often than females, and on average male songs have more notes. Song is influenced by circulating androgens, but how such hormonal differences influence the diverse brain regions involved in vocal display is not understood. To characterize androgen-sensitive sites in the vocal motor pathway, we used two isoforms of pseudorabies virus (PRV) to double-label circuits ending in laryngeal and jaw muscles involved in vocalization, and co-labeled these neurons for androgen receptor (AR). Next we manipulated circulating androgens and observed the effects on AR distribution and male song. We find androgens drive coordinated changes in AR abundance across motor and motivational circuits, and both individual and group differences in song are associated with AR abundance. The results reveal how circulating androgens and the auto-regulation of androgen receptors can influence the diverse circuits necessary for elaborate advertisement displays. Significance statementCourtship and aggressive displays are among those most elaborate and dramatic of sexually dimorphic behaviors. We show that in Alstons singing mouse, an androgen-sensitive network defined by retrograde tracing shapes vocal display effort. Our results reveal how the intensity of singing mouse vocalizations is influenced by androgen actions in the vocal motor pathway.

neuroscience↗

bioRxiv2022

Msh2-Msh3 interferes with DNA metabolism in vivo

Mismatch repair (MMR) is a highly conserved DNA repair pathway that safeguards the genome from errors in DNA replication. In Saccharomyces cerevisiae, two MutS homolog (Msh) complexes, Msh2-Msh3 or Msh2-Msh6, initiate MMR. Msh2-Msh3, the focus of this study, recognizes and directs repair of insertion/deletion loops (IDLs) up to ~17 nucleotides. Msh2-Msh3 also recognizes and binds distinct looped and branched DNA structures with varying affinities, thereby contributing to genome stability outside post-replicative MMR through homologous recombination, double-strand break repair (DSBR), and the DNA damage response. Msh2-Msh3 also promotes genome instability through trinucleotide repeat (TNR) expansions. This non-canonical activity is likely an unfortunate consequence of Msh2-Msh3s intrinsic ability to bind a wide range of DNA structures, including those formed with single-stranded (ss) TNR sequences. We previously demonstrated that Msh2-Msh3 binding to 5 ssDNA flap structures interfered with the in vitro binding and cleavage activities of the flap endonuclease Rad27 (Fen1 in mammals), which promotes 5 ssDNA flap processing during Okazaki fragment maturation (OFM) and long-patch base excision repair (LP-BER). Here we demonstrate that elevated Msh2-Msh3 levels interfere with DNA replication and LP-BER in vivo, consistent with the hypothesis that protein abundance and Msh3 ATPase activities are key drivers of Msh2-Msh3-mediated genomic instability.

genetics↗

bioRxiv2021

An Automated Radio-Telemetry System (ARTS) for Monitoring Small Mammals

Point 1: The study of animals in nature is essential for developing an ecologically valid understanding of behavior. Small mammals, however, are often fossorial and exceedingly difficult to monitor in the wild. This limits both the taxonomic scope of field observation, and excludes species that are powerful models for the study of behavioral mechanisms. Point 2: Here, we implement an automated radio telemetry system (ARTS) designed to track small fossorial mammals. Our ARTS uses an isotropic antenna array coupled with broadband receivers. We characterized transmission at our study site and tested the ARTS ability to track 48 prairie voles. Point 3: We compared position estimates from nonlinear least squares, nonparameteric, and Bayesian trilateration methods and found Bayesian trilateration to have the smallest error. To examine the ability of the system to track biologically significant behavior we used ARTS data to investigate circadian rhythms of freely behaving prairie voles. We used Lomb-Scargle analysis to estimate periodic patterns from irregularly sampled time series of speed. Prairie voles demonstrated ultradian movement at periods of approximately 45 and 90 min, observations on a time scale not possible using data from traditional methods. Point 4: This ARTS offers a new tool to observe rodent field behavior at time scales and in environments which have not been previously possible, such as investigating social and spatial behaviors on the scale of minutes, hours, and days in natural environments.

ecology↗