Target site cleavage by the monomeric restriction enzyme BcnI requires translocation to a random DNA sequence and a switch in enzyme orientation

Endonucleases that generate double-strand breaks in DNA often possess two identical subunits related by rotational symmetry, arranged so that the active sites from each subunit act on opposite DNA strands. In contrast to many endonucleases, Type IIP restriction enzyme BcnI, which recognizes the pseudopalindromic sequence 5'-CCSGG-3' (where S stands for C or G) and cuts both DNA strands after the second C, is a monomer and possesses a single catalytic center. We show here that to generate a double-strand break BcnI nicks one DNA strand, switches its orientation on DNA to match the polarity of the second strand and then cuts the phosphodiester bond on the second DNA strand. Surprisingly, we find that an enzyme flip required for the second DNA strand cleavage occurs without an excursion into bulk solution, as the same BcnI molecule acts processively on both DNA strands. We provide evidence that after cleavage of the first DNA strand, BcnI remains associated with the nicked intermediate and relocates to the opposite strand by a short range diffusive hopping on DNA.     

Degenerate sequence recognition by the monomeric restriction enzyme: single mutation converts BcnI into a strand-specific nicking endonuclease

Unlike orthodox Type II restriction endonucleases that are homodimers and interact with the palindromic 4-8-bp DNA sequences, BcnI is a monomer which has a single active site but cuts both DNA strands within the 5'-CC↓CGG-3'/3'-GGG↓CC-5' target site ('↓' designates the cleavage position). Therefore, after cutting the first strand, the BcnI monomer must re-bind to the target site in the opposite orientation; but in this case, it runs into a different central base because of the broken symmetry of the recognition site. Crystal-structure analysis shows that to accept both the C:G and G:C base pairs at the center of its target site, BcnI employs two symmetrically positioned histidines H77 and H219 that presumably change their protonation state depending on the binding mode. We show here that a single mutation of BcnI H77 or H219 residues restricts the cleavage activity of the enzyme to either the 5'-CCCGG-3' or the 5'-CCGGG-3' strand, thereby converting BcnI into a strand-specific nicking endonuclease. This is a novel approach for engineering of monomeric restriction enzymes into strand-specific nucleases.     

Modulation of the effect of camptothecin in x-irradiated L5178Y-R and L5178Y-S cells by benzamide

The L5178Y (LY) murine lymphoma subline, LY-R, is more radioresistant and more sensitive to camptothecin (CPT, inhibitor of topisomerase I) than the second subline used in our investigation, LY-S. Post-irradiation treatment with 3 μM CPT enhanced the radiosensitivity of LY-S cells (D₀ decrease from 0.52 to 0.34 Gy), but did not change it in LY-R cells. Treatment with 2 mM benzamide [BZ, inhibitor of poly(ADP-ribosylation)] before x-rays and CPT increased the radiosensitivity of LY-R cells (D₀ decrease from 1.15 to 0.52) without further modification of radiosensitivity of LY-S cells. Activity of topoisomerase I was diminished 10 min after x-irradiation (5 Gy) in LY-S, but not in LY-R cells. The data on DNA damage (fluorescent halo or comet assays) showed that the ultimate fate of the cells did not depend on the DNA damage pattern estimated immediately after treatment (e. g. the damage was greater in x-rays plus CPT than in BZ plus x-rays plus CPT treated LY-R cells, although the radiosensitivity was less). Aphidicolin (inhibitor of DNA polymerases α and δ) applied concomitantly with CPT in cells not pre-treated with BZ prevented the increase in DNA damage in LY-R cells, but was without effect in LY-S cells. Taking into account the differential inhibition by x-rays of DNA synthesis in LY sublines and its reversion by BZ in LY-S but not in LY-R cells, we conclude that the pattern of DNA damage observed by the methods applied depended on the status of DNA replication. Received: 28 November 1995 / Accepted in revised form: 20 April 1996     

Composition and biosynthesis of thylakoid membrane polypeptides in the red alga Cyanidium caldarium: Comparison with the thylakoid polypeptide composition of higher plants and cyanobacteria

The polypeptide composition of thylakoid membranes of the red alga Cyanidium caldarium was studied by PAGE in the presence of lithium dodecyl sulfate. The thylakoid membranes were shown to contain 65 polypeptides with mol wt from 110 to 10 kDa. PS I isolated from C. caldarium cells is composed of at least 5 components, one of which is the chlorophyll-protein complex with mol wt of 110 kDa typical of higher plants. Cyt f, c ₅₅₂, b ₆ and b ₅₅₉ were identified. Inhibition of carotenoid biosynthesis with norflurazon caused no changes in the polypeptide composition of thylakoid membranes of the algae grown in dark. The suppression of the biosynthesis rate of some thylakoid polypeptides in the algae grown with norflurazon in light is a result of membrane photodestruction. Thylakoid membranes from C. caldarium cells are more similar in the number of protein components to thylakoid membranes from cells of the cyanobacterium Anacystis nidulans than to those of higher plants (Pisum sativum), which was proved by immune-blotting assays: Thylakoid membranes of the red alga and cyanobacteria contain 28 homologous polypeptides, while thylakoid membranes of the alga and pea, only 15.Abbreviations CD circular dichroism - CP chlorophyll-protein complex - LDS lithium dodecyl sulfate - NF norflurazon     

Proteome–Metabolome Profiling of Ovarian Cancer Ascites Reveals Novel Components Involved in Intercellular Communication

Ovarian cancer ascites is a native medium for cancer cells that allows investigation of their secretome in a natural environment. This medium is of interest as a promising source of potential biomarkers, and also as a medium for cell–cell communication. The aim of this study was to elucidate specific features of the malignant ascites metabolome and proteome. In order to omit components of the systemic response to ascites formation, we compared malignant ascites with cirrhosis ascites. Metabolome analysis revealed 41 components that differed significantly between malignant and cirrhosis ascites. Most of the identified cancer-specific metabolites are known to be important signaling molecules. Proteomic analysis identified 2096 and 1855 proteins in the ovarian cancer and cirrhosis ascites, respectively; 424 proteins were specific for the malignant ascites. Functional analysis of the proteome demonstrated that the major differences between cirrhosis and malignant ascites were observed for the cluster of spliceosomal proteins. Additionally, we demonstrate that several splicing RNAs were exclusively detected in malignant ascites, where they probably existed within protein complexes. This result was confirmed in vitro using an ovarian cancer cell line. Identification of spliceosomal proteins and RNAs in an extracellular medium is of particular interest; the finding suggests that they might play a role in the communication between cancer cells. In addition, malignant ascites contains a high number of exosomes that are known to play an important role in signal transduction. Thus our study reveals the specific features of malignant ascites that are associated with its function as a medium of intercellular communication.Ovarian cancer is the sixth most frequently occurring cancer among the gynecological cancers and accounts for about 5% of all new female cancer cases according to the 2012 data (World Health Organization International Agency for Research on Cancer www.globocan.iarc.fr). Epithelial ovarian cancer registered in 90% of ovarian cancer cases. The rate of mortality from ovarian cancer holds first place among the other gynecological cancers, largely because of the asymptomatic progression of the disease, especially at its early stages, and a lack of adequate screening tests, which leads to late detection, typically only after the cancer has spread to adjacent structures. In such a case, the five-year survival rate is only 25% to 40%, whereas it can be as high as 90% if the cancer is diagnosed early. Unfortunately, ovarian cancer is diagnosed early in less than 20% of the total number of cases (International Agency for Research on Cancer). The main methods for primary diagnostics include transvaginal ultrasound and blood biomarker analyses such as with cancer antigen 125 (CA125),¹ epididymis protein-4 (HE4), and the OVA1 multiparametric (CA125, β2-microglobulin, transferrin, and apolipoprotein A1) tests. The OVA1 test is mainly applied to evaluate the malignancy of a tumor-like pelvic mass, and the other two markers are used to monitor disease progress and estimate treatment efficacy, as they are not highly specific for ovarian cancer and thus produce a high percentage of false-positive results (1). Therefore, the search for specific and sensitive markers for the early diagnosis of ovarian cancer is an urgent problem, although the development of new, efficient methods for treatment of the disease at late stages also remains of critical importance.One of the symptoms associated with late-stage ovarian cancer is excessive fluid accumulation in the abdominal cavity, known as ascites. Mechanisms of malignant ascites formation involve lymph obstruction, activation of mesothelial cells as a result of the metastatic process, and increased vessel permeability due to the secretion of growth factors (2, 3). Therefore, malignant ascites is enriched by tumor cells and soluble growth factors that may be associated with the processes of invasion and metastasis. Thus, ascites provides a native medium for cancer cells and creates an opportunity to investigate the ovarian cancer cell secretome in its natural environment (as distinct from cancer cell cultures in vitro) (2).Omics studies enable us to understand physiological information at different levels (4–7). Considering the highly diverse features of information obtained from each omics platform, one could expect that combinations of different omics should provide highly comprehensive views on special features of the cancer cell secretome. Studies of ascites with the use of omics technologies could not only help us understand the peculiarities of the vital activity of cancer cells in the organism, but also elaborate new therapeutic methods. However, until now, proteomic studies of ovarian cancer ascites have been exceptionally directed at the search for potential biomarkers of this cancer (3, 8–10). Investigation of ascites is also interesting beyond the protein level. In particular, small molecules—metabolites—are known to be involved in intercellular communication. However, in metabolome studies, to our knowledge, metabolites from ovarian cancer ascites have not been explored at all; only metabolomic analysis of urine and serum has been described in the literature for this type of cancer (11–13).It is important to note that ascites accumulation can be caused by various pathologies—for example, liver cirrhosis (81% of all cases), heart diseases (2%), tuberculosis (3%), and 10% of all cases associated with malignancy (10%). The most common cancer associated with ascites is ovarian cancer, accounting for 38% of malignant ascites occurring in females (2). In this study, we compared ascites of different etiologies, formed in the course of ovarian cancer and portal alcoholic cirrhosis. Thus, we not only extended our knowledge of the protein composition and filled in gaps regarding the metabolome, but also elucidated specific features of malignant ascites composition.     

Comparative analysis of proteins selectively released from cell nuclei of different eukaryotic species under conditions of mild hydrolysis by nucleases

Nuclei of cultured Chinese hamster fibroblasts, dechoryonized eggs ofDrosophila melanogaster, calf thymocytes and mouse spleen were digested with micrococcal nuclease or DNAse I under conditions when about 15–20% of nuclear DNA is hydrolyzed. Proteins released from the nuclei with this DNA were analyzed by electrophoresis in polyacrylamide gels in the presence of SDS and the most prominent components were purified in each case. It is shown that these components derived from different species have different electrophoretic mobilities but are very similar in the amino acid composition characterized by high glycine content and a low lysine/arginine ratio. Molecular weights of two of those components, from mouse spleen and thymocytes, were estimated and are shown to be 25,500 and 23,000, respectively. Notwithstanding their different molecular weights they have the same N-terminal amino acid-arginine.