Solution structure of DNA/RNA hybrid duplex with C8-propynyl 2′-deoxyadenosine modifications: Implication of RNase H and DNA/RNA duplex interaction

Solution structures of DNA/RNA hybrid duplexes, d(GCGCA*AA*ACGCG): r(cgcguuuugcg)d(C) (designated PP57), containing two C8-propynyl 2′-deoxyadenosines (A*) and unmodified hybrid (designated U4A4) are solved. The C8-propynyl groups on 2′-deoxyadenosine perturb the local structure of the hybrid duplex, but overall the structure is similar to that of canonical DNA/RNA hybrid duplex except that Hoogsteen hydrogen bondings between A* and U result in lower thermal stability. RNase H is known to cleave RNA only in DNA/RNA hybrid duplexes. Minor groove widths of hybrid duplexes, sugar puckerings of DNA are reported to be responsible for RNase H mediated cleavage, but structural requirements for RNase H mediated cleavage still remain elusive. Despite the presence of bulky propynyl groups of PP57 in the minor groove and greater flexibility, the PP57 is an RNase H substrate. To provide an insight on the interactions between RNase H and substrates we have modeled Bacillus halodurans RNase H-PP57 complex, our NMR structure and modeling study suggest that the residue Gly(15) and Asn(16) of the loop residues between first β sheet and second β sheet of RNase HI of Escherichia coli might participate in substrate binding.     

Isolation and characterization of genes from the marine microalga Pavlova salina encoding three front-end desaturases involved in docosahexaenoic acid biosynthesis

The marine microalga Pavlova salina produces lipids containing approximately 50% omega-3 long chain polyunsaturated fatty acids (LC-PUFA) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Three cDNA sequences, designated PsD4Des, PsD5Des, PsD8Des, were isolated from P. salina and shown to encode three front-end desaturases with Delta4, Delta5 and Delta8 specificity, respectively. Southern analysis indicated that the P. salina genome contained single copies of all three front-end fatty acid desaturase genes. When grown at three different temperatures, analysis of fatty acid profiles indicated P. salina desaturation conversions occurred with greater than 95% efficiency. Real-Time PCR revealed that expression of PsD8Des was higher than for the other two genes under normal growth conditions, while PsD5Des had the lowest expression level. The deduced amino acid sequences from all three genes contained three conserved histidine boxes and a cytochrome b(5) domain. Sequence alignment showed that the three genes were homologous to corresponding desaturases from other microalgae and fungi. The predicted activities of these three front-end desaturases leading to the synthesis of LC-PUFA were also confirmed in yeast and in higher plants.     

Atomistic and coarse-grained analysis of double spectrin repeat units: the molecular origins of flexibility

Spectrin is an ubiquitous protein in metazoan cells, and its flexibility is one of the keys to maintaining cellular structure and organization. Both alpha-spectrin and beta-spectrin polypeptides consist primarily of triple coiled-coil modular repeat units, and two important factors that determine spectrin flexibility are the bending flexibility between two consecutive repeat units and the conformational flexibility of individual repeat units. Atomistic molecular dynamics (MD) simulations are used here to study double spectrin repeat units (DSRUs) from the human erythrocyte beta-spectrin (HEbeta89) and the chicken brain alpha-spectrin (CBalpha1617). From the results of MD simulations, a highly conserved Trp residue in the A-helix of most repeat units that has been suggested to be important in conferring stability to the coiled-coil structures is found not to have a significant effect on the conformational flexibility of individual repeat units. Characterization of the bending flexibility for two consecutive repeats of spectrin via atomistic simulations and coarse-grained (CG) modeling indicate that the bending flexibility is governed by the interactions between the AB-loop of the first repeat unit, the BC-loop of the second repeat unit and the linker region. Specifically, interactions between residues in these regions can lead to a strong directionality in the bending behavior of two repeat units. The biological implications of these finding are discussed.     

An ATP hydrolysis sensor in the DNA packaging motor from bacteriophage T4 suggests an inchworm-type translocation mechanism

Tailed bacteriophages and large eukaryotic viruses employ powerful molecular motors to translocate dsDNA into a preassembled capsid shell. The phage T4 motor is composed of a dodecameric portal and small and large terminase subunits assembled at the special head-tail connector vertex of the prohead. The motor pumps DNA through the portal channel, utilizing ATP hydrolysis energy provided by an ATPase present in the large terminase subunit. We report that the ATPase motors of terminases, helicases, translocating restriction enzymes, and protein translocases possess a common coupling motif (C-motif). Mutations in the phage T4 terminase C-motif lead to loss of stimulated ATPase and DNA translocation activities. Surprisingly, the mutants can catalyze at least one ATP hydrolysis event but are unable to turn over and reset the motor. This is the first report of a catalytic block in translocating ATPase motor after ATP hydrolysis occurred. We suggest that the C-motif is an ATP hydrolysis sensor, linking product release to mechanical motion. A novel terminase-driven mechanism is proposed for translocation of dsDNA in viruses.     

MRN复合物的结构及功能研究进展

MRN复合物包括MRE11、NBS1、RAD50,此复合物中的MRE11或NBS1缺失或突变会导致人的共济失调一毛细血管扩张样疾病、Nijmegen断裂综合征。MRN复合物在DNA双链损伤修复、同源重组、非同源重组、端粒长度维持、细胞检验点激活、保证DNA复制的顺利进行,以及维持基因组的稳定性等方面都起到了重要的作用。从以上几个方面简要综述MRN复合物的研究进展。     

Unity in diversity: Lessons from macaque societies

The macaque radiation is as old as the hominin radiation, approximately 7 million years. After Homo, Macaca has the widest geographical range among primates, and both of these genera are present in tropical and temperate regions as well. Whereas the single extant representative of the genus Homo diverged through processes of cultural diversification, extant species of macaques emerged through processes of evolutionary diversification. Macaque societies are characterized by profound unity and great diversity, and can best be described as variations on the same theme. To understand macaque variation and adaptation, we must take into account the processes that insure the persistence of their societies across generations and environments.     

Effect of replacing [NTf2] by [PF6] anion on the [BMIm][NTf2] ionic liquid confined by gold

The effect of replacing bis(trifluoromethylsulphonyl)imide ([NTf₂]) by hexafluorophosphate ([PF₆]) in room temperature ionic liquid (IL) 1-butyl-3-methylimidazolium bis(trifluoromethylsulphonyl)imide ([BMIm][NTf₂]) confined between two gold interfaces is herein reported through molecular dynamics simulations using all-atom non-polarisable force-fields. Five systems were studied ranging from pure [BMIm][NTf₂] to pure [BMIm][PF₆], with [PF₆] molar fractions of 0, 0.125, 0.25, 0.375 and 0.5. Special attention was drawn to investigate the impact of the [PF₆] anion on the IL, in particular on the first layers of the liquid in close contact with the solid gold surface.     

Ionizing radiation-induced DNA injury and damage detection in patients with breast cancer

Breast cancer is the most common malignancy in women. Radiotherapy is frequently used in patients with breast cancer, but some patients may be more susceptible to ionizing radiation, and increased exposure to radiation sources may be associated to radiation adverse events. This susceptibility may be related to deficiencies in DNA repair mechanisms that are activated after cell-radiation, which causes DNA damage, particularly DNA double strand breaks. Some of these genetic susceptibilities in DNA-repair mechanisms are implicated in the etiology of hereditary breast/ovarian cancer (pathologic mutations in the BRCA 1 and 2 genes), but other less penetrant variants in genes involved in sporadic breast cancer have been described. These same genetic susceptibilities may be involved in negative radiotherapeutic outcomes. For these reasons, it is necessary to implement methods for detecting patients who are susceptible to radiotherapy-related adverse events. This review discusses mechanisms of DNA damage and repair, genes related to these functions, and the diagnosis methods designed and under research for detection of breast cancer patients with increased radiosensitivity.