Quantum chemical studies of structures and binding in noncanonical RNA base pairs: the trans Watson-Crick:Watson-Crick family

The trans Watson-Crick/Watson-Crick family of base pairs represent a geometric class that play important structural and possible functional roles in the ribosome, tRNA, and other functional RNA molecules. They nucleate base triplets and quartets, participate as loop closing terminal base pairs in hair pin motifs and are also responsible for several tertiary interactions that enable sequentially distant regions to interact with each other in RNA molecules. Eleven representative examples spanning nine systems belonging to this geometric family of RNA base pairs, having widely different occurrence statistics in the PDB database, were studied at the HF/6-31G (d, p) level using Morokuma decomposition, Atoms in Molecules as well as Natural Bond Orbital methods in the optimized gas phase geometries and in their crystal structure geometries, respectively. The BSSE and deformation energy corrected interaction energy values for the optimized geometries are compared with the corresponding values in the crystal geometries of the base pairs. For non protonated base pairs in their optimized geometry, these values ranged from -8.19 kcal/mol to -21.84 kcal/mol and compared favorably with those of canonical base pairs. The interaction energies of these base pairs, in their respective crystal geometries, were, however, lesser to varying extents and in one case, that of A:A W:W trans, it was actually found to be positive. The variation in RMSD between the two geometries was also large and ranged from 0.32-2.19 A. Our analysis shows that the hydrogen bonding characteristics and interaction energies obtained, correlated with the nature and type of hydrogen bonds between base pairs; but the occurrence frequencies, interaction energies, and geometric variabilities were conspicuous by the absence of any apparent correlation. Instead, the nature of local interaction energy hyperspace of different base pairs as inferred from the degree of their respective geometric variability could be correlated with the identities of free and bound hydrogen bond donor/acceptor groups present in interacting bases in conjunction with their tertiary and neighboring group interaction potentials in the global context. It also suggests that the concept of isostericity alone may not always determine covariation potentials for base pairs, particularly for those which may be important for RNA dynamics. These considerations are more important than the absolute values of the interaction energies in their respective optimized geometries in rationalizing their occurrences in functional RNAs. They highlight the importance of revising some of the existing DNA based structure analysis approaches and may have significant implications for RNA structure and dynamics, especially in the context of structure prediction algorithms.     

Intrachromosomal homologous recombination in human cells which differ in nucleotide excision-repair capacity

To examine the mechanism of recombination and the role of DNA repair in this process, we transfected a plasmid carrying duplicated copies of the Herpes simplex virus I thymidine kinase (Htk) gene, each containing an 8 bp XhoI site inserted in a unique site and with the neo coding for geneticin resistance located between them, into tk-deficient human cell lines which differ in their ability to carry out nucleotide excision repair. One parental cell line has a normal level of repair activity; the second has an intermediate level, and the third has virtually no repair activity. Several geneticin-resistant transfectant cell strains from each parental line were isolated and assayed for the ability to undergo productive recombination giving rise to tk+ cells. Approximately 25% of them could do so. Southern blot analysis of these transfectants indicated that the majority contained a single copy, or at most, two copies of the plasmid integrated into the chromosome. Fluctuation analysis tests to determine the rate of spontaneous recombination (events per 10(6) cells per cell generation) in the various cell strains showed that the rates ranged from 0.15 to 4.1. The mean rate for the cell strains derived from the repair-deficient cell line was 3.6; for those derived from the cells with an intermediate rate, it was 0.8; and for those with a normal rate of excision repair, it was 0.9. Southern blot analysis of tk+ recombinants showed that in all cases, one of the Htk genes had become wild-type, i.e., XhoI-resistant. 90% of the recombinants retained the Htk gene duplication, consistent with non-reciprocal transfer of genetic information, i.e., gene conversion. The rest contained a single, wild-type Htk gene, consistent with a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids. These cell strains will be useful for investigating the role of DNA damage and repair in homologous recombination.     

Impaired repair activity of a truncated DNA polymerase beta protein.

DNA polymerase beta (polbeta) is an essential enzyme for gap filling synthesis in damaged DNA template involved in base excision repair pathway. A truncated polbeta protein is expressed in primary colorectal and breast adenocarcinomas. To determine a possible alteration in the functions of the enzyme, a human cell line named HeLapolbetadelta expressing the truncated form of polbeta has been established. These cells revealed a significantly reduced level of repair activity evaluated by gap filling synthesis and polbeta activity. More importantly, the HeLapolbetadelta cells are hypersensitive to MNNG, a DNA alkylating agent. It appears from the responses that the gap filling synthesis of WT cells, a HeLa cell line overexpressing wild-type polbeta protein, was inhibited by HeLapolbetadelta protein.     

The role of a heat shock protein from V. cholerae 0139 in the gut immune response

An immunodominant heat shock protein (Hsp 24) was purified from Vibrio cholerae O139 at 42 degrees C and used as an immunomodulator for studying the gut immune response. T cell clone and T cell line specific for the Hsp 24 were generated from the lymphocytes of lamina propria and intra-epithelial lymphocytes of mice orally infected with V. cholerae O139, respectively. The T cell clone was TCR alphabeta(+), CD4(+) and appeared to play an important role in the functioning of gut B-lymphocytes. The T cell line had heterogenous population of CD8+ and CD4+ cells, most of which were found to be TCR alphabeta(+) and a minor population was TCR gammadelta(+). The lymphokine profile of T cell line showed IFN-gamma to be the most abundant lymphokine followed by IL-2 and IL-4. The possible involvement of alternative pathway of activation for T cell clone was also addressed in this study. The splenocytes showed an up-regulation of their CD2 receptor expression on stimulation with the Hsp-24. The pattern of lymphokines released by splenocytes stimulated with the Hsp-24 showed no particular cell type to be responsible for mounting immune response. Thus, there is involvement of both, mucosal and peripheral arm of the immune system.     

Characterization of non-planar peptide groups in protein crystal structures

Peptide groups are generally assumed to be planar in protein structure, due to 'rigid' partial double bond character of peptide bonds, thus the value of peptide torsion angle omega should be restricted to 180 degrees for the usual trans form of peptide unit. However, on analyzing the ultra-high resolution protein crystal database, we find that in some cases, omega deviates >10 degrees from its usual value of 180 degrees, indicating significant non-planarity of peptide groups. Moreover, the non-planarity for most of the amino acids is found to be 'biased' towards values of omega smaller than 180 degrees. Similar trend for to is confirmed by the neutron diffraction data for proteins. The neutron diffraction database also reveals that non-planar peptide groups are generally correlated to 'pyramidal' structure of the peptide-nitrogen bonds. Consequently, the hydrogen atom of peptide group deviates from its planar position, as measured by the 'improper' torsion angle theta. Thus, we find that both the angles omega and theta point towards a significant amount of non-planarity of peptide groups, which cannot be ignored. The role of peptide nonplanarity in protein function is, however, not yet clear.     

Selective impairment of protein kinase C isotypes in murine macrophage by Leismania donovani

Leishmania donovani, an obligate intracellular parasite resides and multiplies within macrophage of the reticuloendothelial system. The intracellular signalling mechanism involved in the impaired oxidative response in leishmaniasis has not yet been clearly established. Generation of superoxide anion (O₂ ^–) is supposed to be the first line of host defence during microbial invasion. We found a substantial inhibition of superoxide anion generation in parasitized macrophages, which was just the reverse in case of macrophages challenged with Lipophosphoglycan (LPG) deficient attenuated leishmanial parasite UR-6. The generation of O₂ ^– essentially needs the prior activation of protein kinase C (PKC) mediated phosphorylation events. Our study proposed that phosphorylation of 67, 54, 47 and 36 kDa proteins was attenuated during infection. This was supported by PKC activity study, where Ca-dependent PKC activity was inhibited but, Ca-independent PKC activity was enhanced. This result was further confirmed by using isotype specific pseudosubstrate inhibitors of Ca-dependent PKC and Ca-independent PKC . Application of -pseudosubstrate could not alter the Ca-dependent PKC activity but -pseudosubstrate inhibited the Ca-independent PKC activity in infected macrophages. Our immunoblot analysis with specific antibody against PKC and PKC isotypes showed down regulation of PKC -II expression with concomitant induction of PKC . Such inhibition of Ca-dependent PKC was reversed in macrophages treated with UR-6. Taken together, our observations revealed that infection with L. donovani selectively attenuates both the expression and activity of Ca-dependent PKC .