Mechanistic insight into the repair of C8-linked pyrrolobenzodiazepine monomer-mediated DNA damage.

Pyrrolobenzodiazepines (PBDs) are naturally occurring DNA binding compounds that possess anti-tumor and anti-bacterial activity. Chemical modifications of PBDs can result in improved DNA binding, sequence specificity and enhanced efficacy. More recently, synthetic PBD monomers have shown promise as payloads for antibody drug conjugates and anti-bacterial agents. The precise mechanism of action of these PBD monomers and their role in causing DNA damage remains to be elucidated. Here we characterized the damage-inducing potential of two C8-linked PBD bi-aryl monomers in Caulobacter crescentus and investigated the strategies employed by cells to repair the same. We show that these compounds cause DNA damage and efficiently kill bacteria, in a manner comparable to the extensively used DNA cross-linking agent mitomycin-C (MMC). However, in stark contrast to MMC which employs a mutagenic lesion tolerance pathway, we implicate essential functions for error-free mechanisms in repairing PBD monomer-mediated damage. We find that survival is severely compromised in cells lacking nucleotide excision repair and to a lesser extent, in cells with impaired recombination-based repair. Loss of nucleotide excision repair leads to significant increase in double-strand breaks, underscoring the critical role of this pathway in mediating repair of PBD-induced DNA lesions. Together, our study provides comprehensive insights into how mono-alkylating DNA-targeting therapeutic compounds like PBD monomers challenge cell growth, and identifies the specific mechanisms employed by the cell to counter the same.

A single center experience of prenatal parent-fetus trio exome sequencing for pregnancies with congenital anomalies.

OBJECTIVES: To examine the diagnostic yield of trio exome sequencing in fetuses with multiple structural defects with no pathogenic findings in cytogenetic and microarray analyses. METHODS: We recruited 51 fetuses with two or more defects, non-immune fetal hydrops or fetal akinesia deformation syndrome|or fetal akinesia deformation sequence (FADS). Trio exome sequencing was performed on DNA from chorionic villi samples and parental blood. Detection of genomic variation and prioritization of clinically relevant variants was performed according to in-house standard operating procedures. RESULTS: Median maternal and gestational age was 32.0 years and 21.0 weeks, respectively. Forty-three (84.3%) fetuses had two or more affected organ systems. The remaining fetuses had isolated fetal hydrops or FADS. In total, the exome analysis established the genetic cause for the clinical abnormalities in 22 (43.1%, 95% CI 29.4%-57.8%) pregnancies. CONCLUSIONS: In fetuses with multiple defects, hydrops or FADS and normal standard genetic results, trio exome sequencing has the potential to identify genetic anomalies in more than 40% of cases.

Diversification of DNA-Binding Specificity by Permissive and Specificity-Switching Mutations in the ParB/Noc Protein Family.

Specific interactions between proteins and DNA are essential to many biological processes. Yet, it remains unclear how the diversification in DNA-binding specificity was brought about, and the mutational paths that led to changes in specificity are unknown. Using a pair of evolutionarily related DNA-binding proteins, each with a different DNA preference (ParB [Partitioning Protein B] and Noc [Nucleoid Occlusion Factor], which both play roles in bacterial chromosome maintenance), we show that specificity is encoded by a set of four residues at the protein-DNA interface. Combining X-ray crystallography and deep mutational scanning of the interface, we suggest that permissive mutations must be introduced before specificity-switching mutations to reprogram specificity and that mutational paths to new specificity do not necessarily involve dual-specificity intermediates. Overall, our results provide insight into the possible evolutionary history of ParB and Noc and, in a broader context, might be useful for understanding the evolution of other classes of DNA-binding proteins.

Clinical and molecular description of 19 patients with GATAD2B-Associated Neurodevelopmental Disorder (GAND).

De novo pathogenic variants in the GATAD2B gene have been associated with a syndromic neurodevelopmental disorder (GAND) characterized by severe intellectual disability (ID), impaired speech, childhood hypotonia, and dysmorphic features. Since its first description in 2013, nine patients have been reported in case reports and a series of 50 patients was recently published, which is consistent with the relative frequency of GATAD2B pathogenic variants in public databases. We report the detailed phenotype of 19 patients from various ethnic backgrounds with confirmed pathogenic GATAD2B variants including intragenic deletions. All individuals presented developmental delay with a median age of 2.5 years for independent walking and of 3 years for first spoken words. GATAD2B variant carriers showed very little subsequent speech progress, two patients over 30 years of age remaining non-verbal. ID was mostly moderate to severe, with one profound and one mild case, which shows a wider spectrum of disease severity than previously reported. We confirm macrocephaly as a major feature in GAND (53%). Most common dysmorphic features included broad forehead, deeply set eyes, hypertelorism, wide nasal base, and pointed chin. Conversely, prenatal abnormalities, non-cerebral malformations, epilepsy, and autistic behavior were uncommon. Other features included feeding difficulties, behavioral abnormalities, and unspecific abnormalities on brain MRI. Improving our knowledge of the clinical phenotype is essential for correct interpretation of the molecular results and accurate patient management.

Sensing and responding to diverse extracellular signals: an updated analysis of the sensor kinases and response regulators of Streptomyces species.

Streptomyces venezuelae is a Gram-positive, filamentous actinomycete with a complex developmental life cycle. Genomic analysis revealed that S. venezuelae encodes a large number of two-component systems (TCSs): these consist of a membrane-bound sensor kinase (SK) and a cognate response regulator (RR). These proteins act together to detect and respond to diverse extracellular signals. Some of these systems have been shown to regulate antimicrobial biosynthesis in Streptomyces species, making them very attractive to researchers. The ability of S. venezuelae to sporulate in both liquid and solid cultures has made it an increasingly popular model organism in which to study these industrially and medically important bacteria. Bioinformatic analysis identified 58 TCS operons in S. venezuelae with an additional 27 orphan SK and 18 orphan RR genes. A broader approach identified 15 of the 58 encoded TCSs to be highly conserved in 93 Streptomyces species for which high-quality and complete genome sequences are available. This review attempts to unify the current work on TCS in the streptomycetes, with an emphasis on S. venezuelae.

Inhibiting the integrated stress response pathway prevents aberrant chondrocyte differentiation thereby alleviating chondrodysplasia.

The integrated stress response (ISR) is activated by diverse forms of cellular stress, including endoplasmic reticulum (ER) stress, and is associated with diseases. However, the molecular mechanism(s) whereby the ISR impacts on differentiation is incompletely understood. Here, we exploited a mouse model of Metaphyseal Chondrodysplasia type Schmid (MCDS) to provide insight into the impact of the ISR on cell fate. We show the protein kinase RNA-like ER kinase (PERK) pathway that mediates preferential synthesis of ATF4 and CHOP, dominates in causing dysplasia by reverting chondrocyte differentiation via ATF4-directed transactivation of Sox9. Chondrocyte survival is enabled, cell autonomously, by CHOP and dual CHOP-ATF4 transactivation of Fgf21. Treatment of mutant mice with a chemical inhibitor of PERK signaling prevents the differentiation defects and ameliorates chondrodysplasia. By preventing aberrant differentiation, titrated inhibition of the ISR emerges as a rationale therapeutic strategy for stress-induced skeletal disorders.

COL10A1 nonsense and frame-shift mutations have a gain-of-function effect on the growth plate in human and mouse metaphyseal chondrodysplasia type Schmid.

Missense, nonsense and frame-shift mutations in the collagen X gene (COL10A1) result in metaphyseal chondrodysplasia type Schmid (MCDS). Complete degradation of mutant COL10A1 mRNA by nonsense-mediated decay in human MCDS cartilage implicates haploinsufficiency in the pathogenesis for nonsense mutations in vivo. However, the mechanism is unclear in situations where the mutant mRNA persist. We show that nonsense/frame-shift mutations can elicit a gain-of-function effect, affecting chondrocyte differentiation in the growth plate. In an MCDS proband, heterozygous for a p.Y663X nonsense mutation, the growth plate cartilage contained 64% wild-type and 36% mutant mRNA and the hypertrophic zone was disorganized and expanded. The in vitro translated mutant collagen X chains, which are truncated, were misfolded, unable to assemble into trimers and interfered with the assembly of normal alpha1(X) chains into trimers. Unlike Col10a1 null mutants, transgenic mice (FCdel) bearing the mouse equivalent of a human MCDS p.P620fsX621 mutation, displayed typical characteristics of MCDS with disproportionate shortening of limbs and early onset coxa vara. In FCdel mice, the degree of expansion of the hypertrophic zones was transgene-dosage dependent, being most severe in mice homozygous for the transgene. Chondrocytes in the lower region of the expanded hypertrophic zone expressed markers uncharacteristic of hypertrophic chondrocytes, indicating that differentiation was disrupted. Misfolded FCdel alpha1(X) chains were retained within the endoplasmic reticulum of hypertrophic chondrocytes, activating the unfolded protein response. Our findings provide strong in vivo evidence for a gain-of-function effect that is linked to the activation of endoplasmic reticulum-stress response and altered chondrocyte differentiation, as a possible molecular pathogenesis for MCDS.