Fused eco29kIR- and M genes coding for a fully functional hybrid polypeptide as a model of molecular evolution of restriction-modification systems.

BACKGROUND: The discovery of restriction endonucleases and modification DNA methyltransferases, key instruments of genetic engineering, opened a new era of molecular biology through development of the recombinant DNA technology. Today, the number of potential proteins assigned to type II restriction enzymes alone is beyond 6000, which probably reflects the high diversity of evolutionary pathways. Here we present experimental evidence that a new type IIC restriction and modification enzymes carrying both activities in a single polypeptide could result from fusion of the appropriate genes from preexisting bipartite restriction-modification systems. RESULTS: Fusion of eco29kIR and M ORFs gave a novel gene encoding for a fully functional hybrid polypeptide that carried both restriction endonuclease and DNA methyltransferase activities. It has been placed into a subclass of type II restriction and modification enzymes--type IIC. Its MTase activity, 80% that of the M.Eco29kI enzyme, remained almost unchanged, while its REase activity decreased by three times, concurrently with changed reaction optima, which presumably can be caused by increased steric hindrance in interaction with the substrate. In vitro the enzyme preferentially cuts DNA, with only a low level of DNA modification detected. In vivo new RMS can provide a 102-fold less protection of host cells against phage invasion. CONCLUSIONS: We propose a molecular mechanism of appearing of type IIC restriction-modification and M.SsoII-related enzymes, as well as other multifunctional proteins. As shown, gene fusion could play an important role in evolution of restriction-modification systems and be responsible for the enzyme subclass interconversion. Based on the proposed approach, hundreds of new type IIC enzymes can be generated using head-to-tail oriented type I, II, and III restriction and modification genes. These bifunctional polypeptides can serve a basis for enzymes with altered recognition specificities. Lastly, this study demonstrates that protein fusion may change biochemical properties of the involved enzymes, thus giving a starting point for their further evolutionary divergence.

New bifunctional restriction-modification enzyme AloI isoschizomer (PcoI): Bioinformatics analysis, purification and activity confirmation.

Type II restriction endonucleases and modification DNA-methyltransferases are key instruments of genetic engineering. Recently the number of proteins assigned to this group exceeds 8500. Subtype IIC organizes bifunctional endonuclease-methyltransferase enzymes and currently consists of 16 described members. Here we present phylogenetic tree of 22 new potential bifunctional endonucleases. The majority of them are thought to be fusions of a restriction nuclease with a DNA-methyltransferase and a target recognition subunit of type I restriction-modification systems (R-M-S structure). A RM.AloI isoschizomer from Prevotella copri DSM-18205, PcoI, has been cloned, purified and its REase activity demonstrated. It cuts DNA in magnesium-dependent manner and demonstrates high affinity to DNA, which probably reflects its mechanism of action. This work provides additional proves that gene fusion might play an important role in evolution of restriction-modification systems and other DNA-modifying proteins.

Two HlyIIR dimers bind to a long perfect inverted repeat in the operator of the hemolysin II gene from Bacillus cereus.

HlyIIR is a negative transcriptional regulator of hemolysin II gene from B. cereus. It binds to a long DNA perfect inverted repeat (44bp) located upstream the hlyII gene. Here we show that HlyIIR is dimeric in solution and in bacterial cells. No protein-protein interactions between dimers and no significant modification of target DNA conformation upon complex formation were observed. Two HlyIIR dimers were found to bind to native operator independently with Kd level in the nanomolar range. The minimal HlyIIR binding site was identified as a half of the long DNA perfect inverted repeat.

Bridge-Induced Translocation between NUP145 and TOP2 Yeast Genes Models the Genetic Fusion between the Human Orthologs Associated With Acute Myeloid Leukemia.

In mammalian organisms liquid tumors such as acute myeloid leukemia (AML) are related to spontaneous chromosomal translocations ensuing in gene fusions. We previously developed a system named bridge-induced translocation (BIT) that allows linking together two different chromosomes exploiting the strong endogenous homologous recombination system of the yeast Saccharomyces cerevisiae. The BIT system generates a heterogeneous population of cells with different aneuploidies and severe aberrant phenotypes reminiscent of a cancerogenic transformation. In this work, thanks to a complex pop-out methodology of the marker used for the selection of translocants, we succeeded by BIT technology to precisely reproduce in yeast the peculiar chromosome translocation that has been associated with AML, characterized by the fusion between the human genes NUP98 and TOP2B. To shed light on the origin of the DNA fragility within NUP98, an extensive analysis of the curvature, bending, thermostability, and B-Z transition aptitude of the breakpoint region of NUP98 and of its yeast ortholog NUP145 has been performed. On this basis, a DNA cassette carrying homologous tails to the two genes was amplified by PCR and allowed the targeted fusion between NUP145 and TOP2, leading to reproduce the chimeric transcript in a diploid strain of S. cerevisiae. The resulting translocated yeast obtained through BIT appears characterized by abnormal spherical bodies of nearly 500 nm of diameter, absence of external membrane and defined cytoplasmic localization. Since Nup98 is a well-known regulator of the post-transcriptional modification of P53 target genes, and P53 mutations are occasionally reported in AML, this translocant yeast strain can be used as a model to test the constitutive expression of human P53. Although the abnormal phenotype of the translocant yeast was never rescued by its expression, an exogenous P53 was recognized to confer increased vitality to the translocants, in spite of its usual and well-documented toxicity to wild-type yeast strains. These results obtained in yeast could provide new grounds for the interpretation of past observations made in leukemic patients indicating a possible involvement of P53 in cell transformation toward AML.

Chromosome translocation may lead to PRK1-dependent anticancer drug resistance in yeast via endocytic actin network deregulation.

Chromosome translocations are often observed in cancer cells, being in some cases the cause of neoplastic transformation while in others the results of it. In previous works, we reproduced this major genomic rearrangement by bridge-induced chromosome translocation (BIT) technology in the model eukaryote Saccharomyces cerevisiae and reported that it affects DNA replication, cell cycle, karyogamy, and cytokinesis while it produces genetic instability. In the present work, we further discovered that this event can lead to increased resistance to anticancer chemicals like Doxorubicin and Latrunculin A via an endocytic actin network deregulation triggered by over-expression of the PRK1 serine/threonine protein kinase gene. This effect is further enhanced by the overexpression of PDR1 and PDR3 transcriptional regulators of pleiotropic drug resistance factors. However, when the actin depolymerizing drug Latrunculin A is forcefully allowed to penetrate through their altered cell wall and membrane barriers, it can kill translocants more efficiently than wild type cells. These observations provide an example of an acquired anticancer drug resistance mechanism and could serve as a lead to how it might be overcome, as any treatment inhibiting genome rearrangements could increase the positive outcome of anticancer therapy by lowering cellular drug resistance.

A new Bacillus cereus DNA-binding protein, HlyIIR, negatively regulates expression of B. cereus haemolysin II.

Haemolysin II, HlyII, is one of several cytotoxic proteins produced by Bacillus cereus, an opportunistic human pathogen that causes food poisoning. The hlyII gene confers haemolytic activity to Escherichia coli cells. Here a new B. cereus gene, hlyIIR, which is located immediately downstream of hlyII and regulates hlyII expression, is reported. The deduced amino acid sequence of HlyIIR is similar to prokaryotic DNA-binding transcriptional regulators of the TetR/AcrA family. Measurements of haemolytic activity levels and of hlyII promoter activity levels using gene fusions and primer-extension assays demonstrated that, in E. coli, hlyII transcription decreased in the presence of hlyIIR. Recombinant HlyIIR binds to a 22 bp inverted DNA repeat centred 48 bp upstream of the hlyII promoter transcription initiation point. In vitro transcription studies showed that HlyIIR inhibits transcription from the hlyII promoter by binding to the 22 bp repeat and RNA polymerase, and by decreasing the formation of the catalytically competent open promoter complex.