Multiple C-Terminal Tails within a Single \u3cem\u3eE. coli\u3c/em\u3e SSB Homotetramer Coordinate DNA Replication and Repair

Escherichia coli single-stranded DNA binding protein (SSB) plays essential roles in DNA replication, recombination and repair. SSB functions as a homotetramer with each subunit possessing a DNA binding domain (OB-fold) and an intrinsically disordered C-terminus, of which the last nine amino acids provide the site for interaction with at least a dozen other proteins that function in DNA metabolism. To examine how many C-termini are needed for SSB function, we engineered covalently linked forms of SSB that possess only one or two C-termini within a four-OB-fold “tetramer”. Whereas E. coli expressing SSB with only two tails can survive, expression of a single-tailed SSB is dominant lethal. E. coli expressing only the two-tailed SSB recovers faster from exposure to DNA damaging agents but accumulates more mutations. A single-tailed SSB shows defects in coupled leading and lagging strand DNA replication and does not support replication restart in vitro. These deficiencies in vitro provide a plausible explanation for the lethality observed in vivo. These results indicate that a single SSB tetramer must interact simultaneously with multiple protein partners during some essential roles in genome maintenance

Reconstitution of a Minimal DNA Replicase From Pseudomonas Aeruginosa and Stimulation by Non-Cognate Auxiliary Factors

DNA polymerase III holoenzyme is responsible for chromosomal replication in bacteria. The components and functions of Escherichia coli DNA polymerase III holoenzyme have been studied extensively. Here, we report the reconstitution of replicase activity by essential components of DNA polymerase holoenzyme from the pathogen Pseudomonas aeruginosa. We have expressed and purified the processivity factor (β), single-stranded DNA-binding protein, a complex containing the polymerase (α) and exonuclease (ϵ) subunits, and the essential components of the DnaX complex (τ3δδ′). Efficient primer elongation requires the presence of αϵ, β, and τ3δδ′. Pseudomonas aeruginosa αϵ can substitute completely for E. coli polymerase III in E. coli holoenzyme reconstitution assays. Pseudomonas β and τ3δδ′ exhibit a 10-fold lower activity relative to their E. coli counterparts in E. coli holoenzyme reconstitution assays. Although the Pseudomonas counterpart to the E. coli ψ subunit was not apparent in sequence similarity searches, addition of purified E. coli χ and ψ (components of the DnaX complex) increases the apparent specific activity of the Pseudomonas τ3δδ′ complex ∼10-fold and enables the reconstituted enzyme to function better under physiological salt conditions

DNA Polymerase III Holoenzyme From Thermus Thermophilus Identification Expression Purification of Components and use to Reconstitute a Processive Replicase

DNA replication in bacteria is performed by a specialized multicomponent replicase, the DNA polymerase III holoenzyme, that consist of three essential components: a polymerase, the β sliding clamp processivity factor, and the DnaX complex clamp-loader. We report here the assembly of the minimal functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme consists of α (pol III catalytic subunit), β (sliding clamp processivity factor), and the essential DnaX (τ/γ), δ and δ′ components of the DnaX complex. We show with purified recombinant proteins that these five components are required for rapid and processive DNA synthesis on long single-stranded DNA templates. Subunit interactions known to occur in DNA polymerase III holoenzyme from mesophilic bacteria including δ-δ′ interaction, δδ′-τ/γ complex formation, and α-τ interaction, also occur within the Tth enzyme. As in mesophilic holoenzymes, in the presence of a primed DNA template, these subunits assemble into a stable initiation complex in an ATP-dependent manner. However, in contrast to replicative polymerases from mesophilic bacteria, Tth holoenzyme is efficient only at temperatures above 50 °C, both with regard to initiation complex formation and processive DNA synthesis. The minimalTth DNA polymerase III holoenzyme displays an elongation rate of 350 bp/s at 72 °C and a processivity of greater than 8.6 kilobases, the length of the template that is fully replicated after a single association event

Discovery and Characterization of the Cryptic Psi Subunit of the Pseudomonad DNA Replicase

We previously reconstituted a minimal DNA replicase from Pseudomonas aeruginosa consisting of α and ϵ (polymerase and editing nuclease), β (processivity factor), and the essential τ, δ, and δ′ components of the clamp loader complex (Jarvis, T., Beaudry, A., Bullard, J., Janjic, N., and McHenry, C. (2005) J. Biol. Chem. 280, 7890-7900). In Escherichia coli DNA polymerase III holoenzyme, χ and Ψ are tightly associated clamp loader accessory subunits. The addition of E. coli χΨ to the minimal P. aeruginosa replicase stimulated its activity, suggesting the existence of χ and Ψ counterparts in P. aeruginosa. The P. aeruginosa χ subunit was recognizable from sequence similarity, but Ψ was not. Here we report purification of an endogenous replication complex from P. aeruginosa. Identification of the components led to the discovery of the cryptic Ψ subunit, encoded by holD. P. aeruginosa χ and Ψ were co-expressed and purified as a 1:1 complex. P. aeruginosa χΨ increased the specific activity of τ3δδ′ 25-fold and enabled the holoenzyme to function under physiological salt conditions. A synergistic effect between χΨ and single-stranded DNA binding protein was observed. Sequence similarity to P. aeruginosa Ψ allowed us to identify Ψ subunits from several other Pseudomonads and to predict probable translational start sites for this protein family. This represents the first identification of a highly divergent branch of the Ψ family and confirms the existence of Ψ in several organisms in which Ψ was not identifiable based on sequence similarity alone

A Three-Domain Structure for the delta Subunit of the DNA Polymerase III Holoenzyme delta Domain III Binds delta\u27 and Assembles Into the DnaX Complex

Using ψ-BLAST, we have developed a method for identifying the poorly conserved δ subunit of the DNA polymerase III holoenzyme from all sequenced bacteria. This approach, starting withEscherichia coli δ, leads not only to the identification of δ but also to the DnaX and δ′ subunits of the DnaX complex and other AAA+-class ATPases. This suggests that, although not an ATPase, δ is related structurally to the other subunits of the DnaX complex that loads the β sliding clamp processivity factor onto DNA. To test this prediction, we aligned δ sequences with those of δ′ and, using the start of δ′ Domain III established from its x-ray crystal structure, predicted the juncture between Domains II and III of δ. This putative δ Domain III could be expressed to high levels, consistent with the prediction that it folds independently. δ Domain III, like Domain III of DnaX and δ′, assembles by itself into a complex with the other DnaX complex components. Cross-linking studies indicated a contact of δ with the DnaX subunits. These observations are consistent with a model where two τ subunits and one each of the γ, δ′, and δ subunits mutually interact to form a pentameric functional core for the DnaX complex

Behavioral, Ecological, and Evolutionary Aspects of Meat-Eating by Sumatran Orangutans (Pongo abelii)

Meat-eating is an important aspect of human evolution, but how meat became a substantial component of the human diet is still poorly understood. Meat-eating in our closest relatives, the great apes, may provide insight into the emergence of this trait, but most existing data are for chimpanzees. We report 3 rare cases of meat-eating of slow lorises, Nycticebus coucang, by 1 Sumatran orangutan mother–infant dyad in Ketambe, Indonesia, to examine how orangutans find slow lorises and share meat. We combine these 3 cases with 2 previous ones to test the hypothesis that slow loris captures by orangutans are seasonal and dependent on fruit availability. We also provide the first (to our knowledge) quantitative data and high-definition video recordings of meat chewing rates by great apes, which we use to estimate the minimum time necessary for a female Australopithecus africanus to reach its daily energy requirements when feeding partially on raw meat. Captures seemed to be opportunistic but orangutans may have used olfactory cues to detect the prey. The mother often rejected meat sharing requests and only the infant initiated meat sharing. Slow loris captures occurred only during low ripe fruit availability, suggesting that meat may represent a filler fallback food for orangutans. Orangutans ate meat more than twice as slowly as chimpanzees (Pan troglodytes), suggesting that group living may function as a meat intake accelerator in hominoids. Using orangutan data as a model, time spent chewing per day would not require an excessive amount of time for our social ancestors (australopithecines and hominids), as long as meat represented no more than a quarter of their diet

The Human Phenotype Ontology in 2024: phenotypes around the world.

The Human Phenotype Ontology (HPO) is a widely used resource that comprehensively organizes and defines the phenotypic features of human disease, enabling computational inference and supporting genomic and phenotypic analyses through semantic similarity and machine learning algorithms. The HPO has widespread applications in clinical diagnostics and translational research, including genomic diagnostics, gene-disease discovery, and cohort analytics. In recent years, groups around the world have developed translations of the HPO from English to other languages, and the HPO browser has been internationalized, allowing users to view HPO term labels and in many cases synonyms and definitions in ten languages in addition to English. Since our last report, a total of 2239 new HPO terms and 49235 new HPO annotations were developed, many in collaboration with external groups in the fields of psychiatry, arthrogryposis, immunology and cardiology. The Medical Action Ontology (MAxO) is a new effort to model treatments and other measures taken for clinical management. Finally, the HPO consortium is contributing to efforts to integrate the HPO and the GA4GH Phenopacket Schema into electronic health records (EHRs) with the goal of more standardized and computable integration of rare disease data in EHRs

Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes