Biology subjects

Gruber, S.

Publications and source records attributed to Gruber, S..

44 records · Page 3Linked to original sources

bioRxiv2020

A rod conformation of the Pyrococcus furiosus Rad50 coiled coil

The Rad50-Mre11 nuclease complex plays a vital role in DNA repair in all domains of life. It recognizes and processes DNA double-strand breaks. Rad50 proteins fold into an extended structure with a ~20-60 nm long coiled coil connecting a globular ABC ATPase domain with a zinc hook dimerization domain. A published structure of an archaeal Rad50 zinc hook shows coiled coils pointing away from each other. Here we present the crystal structure of an alternate conformation displaying co-aligned coiled coils. Archaeal Rad50 may thus switch between rod-shaped and ring-like conformations as recently proposed for a bacterial homolog.

biochemistry↗

bioRxiv2020

Designed Anchoring Geometries Determine Lifetimes of Biotin-Streptavidin Bonds under Constant Load and Enable Ultra-Stable Coupling

The small molecule biotin and the homotetrameric protein streptavidin (SA) form a stable and robust complex that plays a pivotal role in many biotechnological and medical applications. In particular, the biotin-streptavidin linkage is frequently used in single molecule force spectroscopy (SMFS) experiments. Recent data suggest that biotin-streptavidin bonds show strong directional dependence and a broad range of multi-exponential lifetimes under load. Here, we investigate engineered SA variants with different valencies and a unique tethering point under constant forces using a magnetic tweezer assay. We observed two orders-of-magnitude differences in the lifetimes, which we attribute to the distinct force loading geometries in the different SA variants. We identified an especially long-lived tethering geometry that will facilitate ultra-stable SMFS experiments and pave the way for new biotechnological applications.

biophysics↗

bioRxiv2019

Phospho-regulation of the Shugoshin - Condensin interaction at the centromere in budding yeast

Correct bioriented attachment of sister chromatids to mitotic spindle is essential for chromosome segregation. The conserved protein shugoshin (Sgo1) contributes in budding yeast to biorientation by recruiting the protein phosphatase PP2A-Rts1 and the condensin complex to centromeres. Using peptide prints, we identified a Serine-Rich Motif (SRM) of Sgo1 that mediates the interaction with condensin and is essential for centromeric condensin recruitment and the establishment of biorientation. We show that the interaction is regulated via phosphorylation within the SRM and we determined the phospho-sites using mass spectrometry. Analysis of the phosphomimicking and phosphoresistant mutants revealed that SRM phosphorylation disrupts the shugoshin - condensin interaction. We present an evidence that Mps1, a central kinase in the spindle assembly checkpoint, directly phosphorylates Sgo1 within the SRM to regulate the interaction with condensin and thereby condensin localization to centromeres. Our findings identify novel mechanisms that control shugoshin activity at the centromere in budding yeast. Author summaryProper chromosome segregation in eukaryotes is ensured through correct attachment of the spindle microtubules to the centromeric chromosomal regions. The attachment is mediated via the multimolecular proteinaceous complex called kinetochore and precisely regulated. This enables the establishment of so called bioirentation, when each sister chromatid is attached to microtubules emanating from opposite spindle poles. Shugoshin (Sgo1) is a conserved centromeric protein that facilitates biorientation through its interactions with the protein phosphatase PP2A/Rts1, chromosome passanger complex and centromeric condensin. Here, we identified a serin-rich motif that is required for the interaction of shugoshin with the condensin complex. We show that loss of this region impairs condensin enrichment at the centromere, chromosome biorientation, segregation as well as the function of the chromosome passanger complex in the error correction. Moreover, the interaction is phosphoregulated, as phosphorylation of the serin-rich motif on Sgo1 disrupts its interaction with condensin. Finally, we show that the conserved spindle assembly checkpoint kinase Mps1 is responsible for this phosphorylation. Our findings uncover novel regulatory mechanisms that facilitate proper chromosome segregation.

cell biology↗

bioRxiv2019

Spatio-temporal control of DNA replication by the pneumococcal cell cycle regulator CcrZ

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance is poorly understood. Here, we identified a conserved and essential protein in pneumococci and related Firmicutes named CcrZ (for Cell Cycle Regulator protein interacting with FtsZ) that couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae directly interacts with the cytoskeleton protein FtsZ to place it in the middle of the newborn cell where the DnaA-bound origin is positioned. Together, this work uncovers a new mechanism for the control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle.

microbiology↗

bioRxiv2019

Altered White Matter Microstructural Organization in Post-Traumatic Stress Disorder across 3,049 Adults: Results from the PGC-ENIGMA PTSD Consortium

A growing number of studies have examined alterations in white matter organization in people with posttraumatic stress disorder (PTSD) using diffusion MRI (dMRI), but the results have been mixed, which may be partially due to relatively small sample sizes among studies. Altered structural connectivity may be both a neurobiological vulnerability for, and a result of, PTSD. In an effort to find reliable effects, we present a multi-cohort analysis of dMRI metrics across 3,049 individuals from 28 cohorts currently participating in the PGC-ENIGMA PTSD working group (a joint partnership between the Psychiatric Genomics Consortium and the Enhancing NeuroImaging Genetics through Meta-Analysis consortium). Comparing regional white matter metrics across the full brain in 1,446 individuals with PTSD and 1,603 controls (2152 males/897 females) between ages 18-83, 92% of whom were trauma-exposed, we report associations between PTSD and disrupted white matter organization measured by lower fractional anisotropy (FA) in the tapetum region of the corpus callosum (Cohens d=-0.12, p=0.0021). The tapetum connects the left and right hippocampus, structures for which structure and function have been consistently implicated in PTSD. Results remained significant/similar after accounting for the effects of multiple potentially confounding variables: childhood trauma exposure, comorbid depression, history of traumatic brain injury, current alcohol abuse or dependence, and current use of psychotropic medications. Our results show that PTSD may be associated with alterations in the broader hippocampal network.

neuroscience↗

bioRxiv2018

Separate Compartments for Chromosome Entrapment and DNA Binding during SMC translocation

Multi-subunit SMC ATPase complexes translocate on chromosomal DNA. They control chromosome structure and DNA topology, presumably by acting as DNA extrusion motors. The SMC-kleisin ring entraps chromosomal DNA. The ring lumen is strongly reduced in size by alignment of the SMC arms and upon ATP binding is divided in two by engagement of SMC head domains. Here, we provide evidence for DNA binding in the SMC compartment and chromosome entrapment in the Kleisin compartment of B. subtilis Smc/ScpAB. We show that DNA binding at the Smc hinge is dispensable and identify an essential DNA binding site at engaged heads which faces the S compartment. Mutations interfering with DNA binding do not prevent ATP hydrolysis but block DNA translocation by Smc/ScpAB. Our findings are consistent with the notion that Smc/DNA contacts stabilize looped DNA segments in the S compartment, while the base of a chromosomal DNA loop is enclosed in the K compartment. Transfer of DNA double helices between S and K compartments may support DNA translocation.

biochemistry↗

bioRxiv2018

DNA-segment-capture model for loop extrusion by structural maintenance of chromosome (SMC) protein complexes

Cells possess remarkable control of the folding and entanglement topology of long and flexible chromosomal DNA molecules. It is thought that structural maintenance of chromosome (SMC) protein complexes play a crucial role in this, by organizing long DNAs into series of loops. Recent experimental data suggest that SMC complexes are able to translocate on DNA, as well as pull out lengths of DNA via a \"loop extrusion\" process. We describe a Brownian loop-capture-ratchet model for translocation and loop extrusion based on known structural, catalytic, and DNA-binding properties of the Bacillus subtilis SMC complex. Our model provides an example of a new class of molecular motor where large conformational fluctuations of the motor track - in this case DNA - are involved in the basic translocation process. Quantitative analysis of our model leads to a series of predictions for the motor properties of SMC complexes, most strikingly a strong dependence of SMC translocation velocity on tension in the DNA track that it is moving along, with \"stalling\" occuring at subpiconewton tensions. A closely related hallmark of the loop-capture mechanism is a strong dependence of translocation step size on DNA tension. We also discuss how the same mechanism might be used by other structurally related SMC complexes (E. coli MukBEF and eukaryote condensin, cohesin and SMC5/6) to organize genomic DNA.

biophysics↗

bioRxiv2018

Temperature-dependent estimation of Gibbs energies using an updated group contribution method

Reaction equilibrium constants determine the mass action ratios necessary to drive flux through metabolic pathways. Group contribution methods offer a way to estimate reaction equilibrium constants at wide coverage across the metabolic network. Here, we present an updated group contribution method with: 1) additional curated thermodynamic data used in fitting; and 2) capabilities to calculate equilibrium constants as a function of temperature. We first collected and curated aqueous thermo-dynamic data, including reaction equilibrium constants, enthalpies of reaction, Gibbs free energies of formation, enthalpies of formation, entropies change of formation of compounds, and proton and metal ion binding constants. We further estimated magnesium binding constants for 618 compounds using a linear regression model validated against measured data. Next, we formulated the calculation of equilibrium constants as a function of temperature and calculated necessary parameters, including standard entropy change of formation ({Delta}f S{circ}) and standard entropy change of reaction ({Delta}rS{circ}), using a model based on molecular properties. The median absolute errors in estimating {Delta}f S{circ} and {Delta}rS{circ} were 0.010 kJ/K/mol and 0.018 kJ/K/mol, respectively. The efforts here fill in gaps for thermodynamic calculations under various conditions, specifically different temperatures and metal ion concentrations. These results support the study of thermodynamic driving forces underlying the metabolic function of organisms living under diverse conditions.

biophysics↗