The MCM-Binding Protein ETG1 Aids Sister Chromatid Cohesion Required for Postreplicative Homologous Recombination Repair

The DNA replication process represents a source of DNA stress that causes potentially spontaneous genome damage. This effect might be strengthened by mutations in crucial replication factors, requiring the activation of DNA damage checkpoints to enable DNA repair before anaphase onset. Here, we demonstrate that depletion of the evolutionarily conserved minichromosome maintenance helicase-binding protein ETG1 of Arabidopsis thaliana resulted in a stringent late G2 cell cycle arrest. This arrest correlated with a partial loss of sister chromatid cohesion. The lack-of-cohesion phenotype was intensified in plants without functional CTF18, a replication fork factor needed for cohesion establishment. The synergistic effect of the etg1 and ctf18 mutants on sister chromatid cohesion strengthened the impact on plant growth of the replication stress caused by ETG1 deficiency because of inefficient DNA repair. We conclude that the ETG1 replication factor is required for efficient cohesion and that cohesion establishment is essential for proper development of plants suffering from endogenous DNA stress. Cohesion defects observed upon knockdown of its human counterpart suggest an equally important developmental role for the orthologous mammalian ETG1 protein.     

Patterns of transmission and horizontal gene transfer in the Dioscorea sansibarensis leaf symbiosis revealed by whole-genome sequencing

1. Download : Download high-res image (221KB)
2. Download : Download full-size image

     

Molecular identification of β-citrylglutamate hydrolase as glutamate carboxypeptidase 3

β-Citrylglutamate (BCG), a compound present in adult testis and in the CNS during the pre- and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify β-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca(2+), the purified enzyme specifically hydrolyzed β-citrylglutamate and did not act on N-acetyl-aspartylglutamate (NAAG). However, both compounds were hydrolyzed in the presence of Mn(2+). This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that β-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of N-acetyl-aspartylglutamate. The mouse tissue distribution of β-citrylglutamate hydrolase was strikingly similar to that of the glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to β-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed β-citrylglutamate in the presence of Ca(2+), and acted on both N-acetyl-aspartylglutamate and β-citrylglutamate in the presence of Mn(2+), whereas recombinant GCP2 only hydrolyzed N-acetyl-aspartylglutamate and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis experiments revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) is largely responsible for GCP3 being able to hydrolyze β-citrylglutamate. Based on the crystal structure of GCP3 and kinetic analysis, we propose that GCP3 forms a labile catalytic Zn-Ca cluster that is critical for its β-citrylglutamate hydrolase activity.     

The plasticity of the β-trefoil fold constitutes an evolutionary platform for protease inhibition

Proteases carry out a number of crucial functions inside and outside the cell. To protect the cells against the potentially lethal activities of these enzymes, specific inhibitors are produced to tightly regulate the protease activity. Independent reports suggest that the Kunitz-soybean trypsin inhibitor (STI) family has the potential to inhibit proteases with different specificities. In this study, we use a combination of biophysical methods to define the structural basis of the interaction of papaya protease inhibitor (PPI) with serine proteases. We show that PPI is a multiple-headed inhibitor; a single PPI molecule can bind two trypsin units at the same time. Based on sequence and structural analysis, we hypothesize that the inherent plasticity of the β-trefoil fold is paramount in the functional evolution of this family toward multiple protease inhibition.     

Genotoxicity investigation of ELF-magnetic fields in Salmonella typhimurium with the sensitive SOS-based VITOTOX test

We performed a genotoxicity investigation of extremely low-frequency (ELF) magnetic fields (MFs, 50 Hz, 100 and 500 μT, 1 and 2 h exposure) alone and in combination with known chemical mutagens using the VITOTOX test. This test is a very sensitive reporter assay of Salmonella typhimurium bacteria based on the SOS response. Our study showed that ELF-MFs do not induce SOS-based mutagenicity in S. typhimurium bacteria and do not show any synergetic effect when combined with chemical mutagens.     

Effects of gamma-ray-induced free radicals on the metal content and amino acid composition of human metallothionein-1

Metallothioneins (MTs), a low-mass class of metalloproteins, are characterized by a high thiolate sulphur and metal content. MTs are involved in metal homeostasis and heavy metal detoxification, and are efficient scavengers of free radicals. This article describes zinc release from human MT-1 and modification of its amino acid composition when subjected to free radicals generated during gamma ray radiolysis. The effect of gamma ray radiolysis of untreated and metal-depleted human MT-1 was tested under multiple aerobic and anaerobic conditions at increasing irradiation doses. Under all conditions, a rapid increase of serine in the early stages of irradiation was observed. Irradiation for longer times led to cysteic acid formation, except under argon atmosphere. Several other amino acid concentrations gradually decreased. Formation of limited amounts of hydroxyproline, hydroxylysine and ornithine as well as some less common derivatives such as cystathionine occurred as side-effects.     

Arcobacter population dynamics in pigs on farrow-to-finish farms

Healthy pigs are an important reservoir for the emerging human pathogen Arcobacter which can result in contamination of porcine carcasses and pork and the spread of arcobacters into the environment. Up to now, the excretion of arcobacters by pigs has been studied, but information about the transmission routes in fattening pigs is lacking. The present study aimed to elucidate the Arcobacter population dynamics in pigs during the fattening period on four farrow-to-finish farms. On each farm, 30 clinically healthy, 12-week-old piglets were selected. Fecal samples were collected on 10 sampling occasions until a slaughter age of 30 weeks was reached. Arcobacter spp. were isolated by a selective method and identified by multiplex PCR. The genetic diversity was examined by amplified fragment length polymorphism and enterobacterial repetitive intergenic consensus PCR. The Arcobacter presence in the fecal samples on the four farms ranged from 11.3 to 50.0%, with excretion levels of up to 10(4) CFU/g feces. The ratio in which Arcobacter species were isolated varied between the farms and over time. Characterization revealed a high degree of genotypic diversity among the isolates. Arcobacter strains persisted and spread within the finishing unit during the fattening period. The occurrence of both unique and shared genotypes in pigs in adjacent and nonadjacent pens demonstrates that transmission routes other than fecal-oral transmission occur.