Challenging a Paradigm: the Role of DNA Homology in Tyrosine Recombinase Reactions

Summary: A classical feature of the tyrosine recombinase family of proteins catalyzing site-specific recombination, as exemplified by the phage lambda integrase and the Cre and Flp recombinases, is the ability to recombine substrates sharing very limited DNA sequence identity. Decades of research have established the importance of this short stretch of identity within the core regions of the substrates. Since then, several new enzymes that challenge this paradigm have been discovered and require the role of sequence identity in site-specific recombination to be reconsidered. The integrases of the conjugative transposons such as Tn916, Tn1545, and CTnDOT recombine substrates with heterologous core sequences. The integrase of the mobilizable transposon NBU1 performs recombination more efficiently with certain core mismatches. The integration of CTX phage and capture of gene cassettes by integrons also occur by altered mechanisms. In these systems, recombination occurs between mismatched sequences by a single strand exchange. In this review, we discuss literature that led to the formulation of the current strand-swapping isomerization model for tyrosine recombinases. The review then focuses on recent developments on the recombinases that challenged the paradigm that was derived from the studies of early systems.     

The phosphoramidate ProTide approach greatly enhances the activity of β-2′-C-methylguanosine against hepatitis C virus

β-2′-C-Methyl purines (1, 2) are known inhibitors of hepatitis C virus (HCV). We herein report the synthesis, biological and enzymatic evaluation of their 5′-phosphoramidate ProTides. Described herein are seven l-alanine phosphoramidate derivatives with variations to the amino acid ester. The 1-naphthyl phosphoramidate of β-2′-methylguanosine containing the benzyl ester (20) was the most active at 0.12 μM, an 84-fold of increase in activity compared to the parent nucleoside (2) with no increase of cytotoxicity. The carboxypeptidase mediated hydrolysis of several ProTides showed a predictive correlation with their activity versus HCV in replicon.     

Synthesis and NMR properties of novel 5,6-dihydroborauracil derivatives

Novel boron compounds - 5,6-saturated borauracil derivatives (4-bromo-5,6-dihydroborauracil, 4-hydroxy-5,6-dihydroborauracil and 4-methoxy-5,6-dihydroborauracil) are presented along with other boron compounds obtained from N-vinylurea: N-substituted β-boronic amino acid - 2-{[(dihydroxyborano-amino)(dihydroxyboranooxy)methyl]-amino}ethylboronic acid and substituted methoxy-borane O-[(1-amino-1-N-vinylamino)methyl]dihydroxyboronate.     

Synthesis and NMR properties of derivatives of 5,6-dihydroborauracil and 5,6-dihydroborathymine

Novel boron compounds, a series of 4-hydroxy-5,6-dihydroborauracil and 4-hydroxy-5,6-dihydroborathymine derivatives containing various substituents at 3-, 5- and 6-positions, is presented. The spectroscopic properties, along with analyses of NMR-controlled boron compound–alcohol and boron compound–amine interactions, proves the existence of sp³-hybridized, stable B,B-bis-methoxy-5,6-dihydroborauracils and pyridine-/n-butylamine-5,6-dihydroborauracils ate-complexes in solution.     

Quantification of Normal Cell Fraction and Copy Number Neutral LOH in Clinical Lung Cancer Samples Using SNP Array Data

Background

Technologies based on DNA microarrays have the potential to provide detailed information on genomic aberrations in tumor cells. In practice a major obstacle for quantitative detection of aberrations is the heterogeneity of clinical tumor tissue. Since tumor tissue invariably contains genetically normal stromal cells, this may lead to a failure to detect aberrations in the tumor cells.

Principal Finding

Using SNP array data from 44 non-small cell lung cancer samples we have developed a bioinformatic algorithm that accurately models the fractions of normal and tumor cells in clinical tumor samples. The proportion of normal cells in combination with SNP array data can be used to detect and quantify copy number neutral loss-of-heterozygosity (CNNLOH) in the tumor cells both in crude tumor tissue and in samples enriched for tumor cells by laser capture microdissection.

Conclusion

Genome-wide quantitative analysis of CNNLOH using the CNNLOH Quantifier method can help to identify recurrent aberrations contributing to tumor development in clinical tumor samples. In addition, SNP-array based analysis of CNNLOH may become important for detection of aberrations that can be used for diagnostic and prognostic purposes.     

Potassium channels in the mitochondria of unicellular eukaryotes and plants

The functional characterisation of potassium channels found in the mitochondria of plants and unicellular eukaryotes is critically discussed herein, with a focus on the ATP-sensitive potassium channel and the large-conductance Ca²⁺-activated potassium channel (mitoBK_Ca channel). The physiological functions of these channels are not completely understood. We discuss the functional connections and roles of potassium channels, uncoupling protein and alternative oxidase, three energy-dissipating systems that exist in the mitochondrial respiratory chain of plants and some unicellular eukaryotes, which include preventing the production of reactive oxygen species.     

New archaeal methyltransferases forming 1-methyladenosine or 1-methyladenosine and 1-methylguanosine at position 9 of tRNA

Two archaeal tRNA methyltransferases belonging to the SPOUT superfamily and displaying unexpected activities are identified. These enzymes are orthologous to the yeast Trm10p methyltransferase, which catalyses the formation of 1-methylguanosine at position 9 of tRNA. In contrast, the Trm10p orthologue from the crenarchaeon Sulfolobus acidocaldarius forms 1-methyladenosine at the same position. Even more surprisingly, the Trm10p orthologue from the euryarchaeon Thermococcus kodakaraensis methylates the N¹-atom of either adenosine or guanosine at position 9 in different tRNAs. This is to our knowledge the first example of a tRNA methyltransferase with a broadened nucleoside recognition capability. The evolution of tRNA methyltransferases methylating the N¹ atom of a purine residue is discussed.     

Bacteria Penetrate the Inner Mucus Layer before Inflammation in the Dextran Sulfate Colitis Model

Background

Protection of the large intestine with its enormous amount of commensal bacteria is a challenge that became easier to understand when we recently could describe that colon has an inner attached mucus layer devoid of bacteria (Johansson et al. (2008) Proc. Natl. Acad. Sci. USA 105, 15064–15069). The bacteria are thus kept at a distance from the epithelial cells and lack of this layer, as in Muc2-null mice, allow bacteria to contact the epithelium. This causes colitis and later on colon cancer, similar to the human disease Ulcerative Colitis, a disease that still lacks a pathogenetic explanation. Dextran Sulfate (DSS) in the drinking water is the most widely used animal model for experimental colitis. In this model, the inflammation is observed after 3–5 days, but early events explaining why DSS causes this has not been described.

Principal Findings

When mucus formed on top of colon explant cultures were exposed to 3% DSS, the thickness of the inner mucus layer decreased and became permeable to 2 µm fluorescent beads after 15 min. Both DSS and Dextran readily penetrated the mucus, but Dextran had no effect on thickness or permeability. When DSS was given in the drinking water to mice and the colon was stained for bacteria and the Muc2 mucin, bacteria were shown to penetrate the inner mucus layer and reach the epithelial cells already within 12 hours, long before any infiltration of inflammatory cells.

Conclusion

DSS thus causes quick alterations in the inner colon mucus layer that makes it permeable to bacteria. The bacteria that reach the epithelial cells probably trigger an inflammatory reaction. These observations suggest that altered properties or lack of the inner colon mucus layer may be an initial event in the development of colitis.     

The suppressor domain of inositol 1,4,5-trisphosphate receptor plays an essential role in the protection against apoptosis

The N-terminal 1-225 amino acids (aa) of the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) function as a suppressor/coupling domain. In this study we used IP(3)R-deficient B-lymphocytes to investigate the effects of modifications in this domain on IP(3) binding and Ca(2+)-release activity. Although the N-terminal 1-225 aa of IP(3)R3 had the same role as in IP(3)R1, the suppression of IP(3) binding for IP(3)R1 was lost when the suppressor/coupling domains were exchanged between the two isoforms. Resulting chimeric receptors showed a higher sensitivity to IP(3)-induced activation (IICR). Deletion of 11 aa in IP(3)R1 ([Delta76-86]-IP(3)R1) or replacing aa 76-86 of the IP(3)R1 in the suppressor/coupling domain by 13 aa of IP(3)R3 ([75-87 T3]-IP(3)R1) also resulted in increased IP(3) binding and sensitivity of IICR. These residues constitute the only part of the suppressor/coupling domain that is strikingly different between the two isoforms. Expression of [Delta76-86]-IP(3)R1 and of [75-87 T3]-IP(3)R1 increased the propensity of cells to undergo staurosporine-induced apoptosis, but had no effect on the Ca(2+) content in the endoplasmic reticulum. In the cell model used, our observations suggest that the sensitivity of the Ca(2+)-release activity of IP(3)R1 to IP(3) influences the sensitivity of the cells to apoptotic stimuli and that the suppressor/coupling domain may have an anti-apoptotic function by attenuating the sensitivity of IICR.