The dynamic nature of the human origin recognition complex revealed through five cryoEM structures

Genome replication is initiated from specific origin sites established by dynamic events. The Origin Recognition Complex (ORC) is necessary for orchestrating the initiation process by binding to origin DNA, recruiting CDC6, and assembling the MCM replicative helicase on DNA. Here we report five cryoEM structures of the human ORC (HsORC) that illustrate the native flexibility of the complex. The absence of ORC1 revealed a compact, stable complex of ORC2-5. Introduction of ORC1 opens the complex into several dynamic conformations. Two structures revealed dynamic movements of the ORC1 AAA+ and ORC2 winged-helix domains that likely impact DNA incorporation into the ORC core. Additional twist and pinch motions were observed in an open ORC conformation revealing a hinge at the ORC5·ORC3 interface that may facilitate ORC binding to DNA. Finally, a structure of ORC was determined with endogenous DNA bound in the core revealing important differences between human and yeast origin recognition.

The dynamic nature of the human Origin Recognition Complex revealed through five cryoEM structures

Genome replication is initiated from specific origin sites established by dynamic events. The Origin Recognition Complex (ORC) is necessary for orchestrating the initiation process by binding to origin DNA, recruiting CDC6, and assembling the MCM replicative helicase on DNA. Here we report five cryoEM structures of the human ORC (HsORC) that illustrate the native flexibility of the complex. The absence of ORC1 revealed a compact, stable complex of ORC2-5. Introduction of ORC1 opens the complex into several dynamic conformations. Two structures revealed dynamic movements of the ORC1 AAA+ and ORC2 winged-helix domains that likely impact DNA incorporation into the ORC core. Additional twist and pinch motions were observed in an open ORC conformation revealing a hinge at the ORC5·3 interface that may facilitate ORC binding to DNA. Finally, a structure of ORC was determined with endogenous DNA bound in the core revealing important differences between human and yeast origin recognition.

Stable mutants of restriction-deficient/modification-proficient Bacillus subtilis 168: hub strains for giant DNA engineering

Bacillus subtilis 168 has been explored as a platform for the synthesis and transmission of large DNA. Two inherent DNA incorporation systems, natural transformation and pLS20-based conjugation transfer, enable rapid handling of target DNA. Both systems are affected by the Bsu restriction–modification system that recognizes and cleaves unmethylated XhoI sites, limiting the choice of target DNA. We constructed B. subtilis 168 with stable mutation for restriction-deficient and modification-proficient (r−m+). It was demonstrated that the r−m+ strains can incorporate and transfer synthesized DNA with multiple XhoI sites. These should be of value as hub strains to integrate and disseminate giant DNA between B. subtilis 168 derivatives.

Acrylamide-dT: a polymerisable nucleoside for DNA incorporation

Nucleoside derivative Acrylamide-dT can be readily synthesised and incorporated multiple times into DNA sequences at any position via automated synthesis.

Study of the incorporation of nucleic acids in chitosan-coated polystyrene nanoparticles for use as DNA carrier system

Nanotechnology is a multidisciplinary scientific field based on the development, characterization, production and application of structures, devices and systems with shape and size at the nanoscale. Polymeric systems with therapeutic purpose have been widely used since they allow a slow and gradual release of drug and allow the transport of drugs to their specific place of action. In recent years, nanoparticles have been used for DNA loading. The introduction of exogenous DNA into a cell may be applicable to fields of gene therapy, DNA vaccines and diagnosis. The development of nucleic acid loading nanoparticles, with a well characterized activity, would be very important. For this project, cationic polystyrene nanoparticles coated with chitosan was studied for a DNA carrier system. The propose is an elaboration of a dilution gradient that allows to know the pattern of incorporation of nucleic acids in the nanoparticles, permitting the development of a mathematical model that characterizes the incorporation in the different conditions studied, allowing their use in future projects. Through this, it´s found the potential of DNA saturation by this nanoparticle system, as in 29% of the incorporation mass, which reveals the capacity of DNA incorporation.

Comparative analysis based on replication banding reveals the mechanism responsible for the difference in the karyotype constitution of treefrogs Ololygon and Scinax (Arboranae, Hylidae, Scinaxinae)

According to the recent taxonomic and phylogenetic revision of the family Hylidae, species of the former Scinaxcatharinae (Boulenger, 1888) clade were included in the resurrected genus Ololygon Fitzinger, 1843, while species of the Scinaxruber (Laurenti, 1768) clade were mostly included in the genus Scinax Wagler, 1830, and two were allocated to the newly created genus JulianusDuellman et al., 2016. Although all the species of the former Scinax genus shared a diploid number of 2n = 24 and the same fundamental number of chromosome arms of FN = 48, two karyotypic constitutions were unequivocally recognized, related mainly to the distinct size and morphology of the first two chromosome pairs. Some possible mechanisms for these differences had been suggested, but without any experimental evidence. In this paper, a comparison was carried out based on replication chromosome banding, obtained after DNA incorporation of 5-bromodeoxiuridine in chromosomes of Ololygon and Scinax. The obtained results revealed that the loss of repetitive segments in chromosome pairs 1 and 2 was the mechanism responsible for karyotype difference. The distinct localization of the nucleolus organizer regions in the species of both genera also differentiates the two karyotypic constitutions.

Comparative analysis based on replication banding reveals the mechanism responsible for the difference in the karyotype constitution of treefrogs Ololygon and Scinax (Arboranae, Hylidae, Scinaxinae)

According to the recent taxonomic and phylogenetic revision of the family Hylidae, species of the former Scinaxcatharinae (Boulenger, 1888) clade were included in the resurrected genus Ololygon Fitzinger, 1843, while species of the Scinaxruber (Laurenti, 1768) clade were mostly included in the genus Scinax Wagler, 1830, and two were allocated to the newly created genus JulianusDuellman et al., 2016. Although all the species of the former Scinax genus shared a diploid number of 2n = 24 and the same fundamental number of chromosome arms of FN = 48, two karyotypic constitutions were unequivocally recognized, related mainly to the distinct size and morphology of the first two chromosome pairs. Some possible mechanisms for these differences had been suggested, but without any experimental evidence. In this paper, a comparison was carried out based on replication chromosome banding, obtained after DNA incorporation of 5-bromodeoxiuridine in chromosomes of Ololygon and Scinax. The obtained results revealed that the loss of repetitive segments in chromosome pairs 1 and 2 was the mechanism responsible for karyotype difference. The distinct localization of the nucleolus organizer regions in the species of both genera also differentiates the two karyotypic constitutions.