Dna2p helicase/nuclease is a tracking protein, like FEN1, for flap cleavage during Okazaki fragment maturation

During cellular DNA replication the lagging strand is generated as discontinuous segments called Okazaki fragments. Each contains an initiator RNA primer that is removed prior to joining of the strands. Primer removal in eukaryotes requires displacement of the primer into a flap that is cleaved off by flap endonuclease 1 (FEN1). FEN1 employs a unique tracking mechanism that requires the recognition of the free 5' terminus and then movement to the base of the flap for cleavage. Abnormally long flaps are coated by replication protein A (RPA), inhibiting FEN1 cleavage. A second nuclease, Dna2p, is needed to cleave an RPA-coated flap producing a short RPA-free flap, favored by FEN1. Here we show that Dna2p is also a tracking protein. Annealed primers or conjugated biotin-streptavidin complex block Dna2p entry and movement. Single-stranded binding protein-coated flaps inhibit Dna2p cleavage. Like FEN1, Dna2p can track over substrates with a non-Watson Crick base, such as a biotin, or a missing base within a chain. Unlike FEN1, Dna2p shows evidence of a "threading-like" mechanism that does not support tracking over a branched substrate. We propose that the two nucleases both track, Dna2p first and then FEN1, to remove initiator RNA via long flap intermediates.     

Stability of the allergenic soybean Kunitz trypsin inhibitor

The soybean Kunitz trypsin inhibitor (SKTI) is a 21.5 kDa allergenic protein that belongs to the family of all antiparallel beta-sheet proteins that are highly resistant to thermal and chemical denaturation. Spectroscopic and biochemical techniques such as circular dichroism (CD), ANS fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid (SGF). Limited proteolysis with chymotrypsin during renaturation after heating showed that the native structure reforms at around 60 degrees C reversing the denaturation. CD spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten structure. The existence of this intermediate was established by ANS fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid denaturation may be important for its role as a food allergen.     

The Plant Host Can Affect the Encapsidation of Brome Mosaic Virus (BMV) RNA: BMV Virions Are Surprisingly Heterogeneous

Brome mosaic virus (BMV) packages its genomic and subgenomic RNAs into three separate viral particles. BMV purified from barley, wheat, and tobacco have distinct relative abundances of the encapsidated RNAs. We seek to identify the basis for the host-dependent differences in viral RNA encapsidation. Sequencing of the viral RNAs revealed recombination events in the 3′ untranslated region of RNA1 of BMV purified from barley and wheat, but not from tobacco. However, the relative amounts of the BMV RNAs that accumulated in barley and wheat are similar and RNA accumulation is not sufficient to account for the difference in RNA encapsidation. Virions purified from barley and wheat were found to differ in their isoelectric points, resistance to proteolysis, and contacts between the capsid residues and the RNA. Mass spectrometric analyses revealed that virions from the three hosts had different post-translational modifications that should impact the physiochemical properties of the virions. Another major source of variation in RNA encapsidation was due to the purification of BMV particles to homogeneity. Highly enriched BMV present in lysates had a surprising range of sizes, buoyant densities, and distinct relative amounts of encapsidated RNAs. These results show that the encapsidated BMV RNAs reflect a combination of host effects on the physiochemical properties of the viral capsids and the enrichment of a subset of virions. The previously unexpected heterogeneity in BMV should influence the timing of the infection and also the host innate immune responses.     

Viscolin, a new chalcone from Viscum coloratum, inhibits human neutrophil superoxide anion and elastase release via a cAMP-dependent pathway

The mistletoe Viscum coloratum is used in traditional Chinese medicine to treat inflammatory diseases. In this study, a cellular model in isolated human neutrophils, which are important in the pathogenesis of rheumatoid arthritis, chronic obstructive pulmonary disease, and other inflammatory diseases, was established to elucidate the anti-inflammatory functions of V. coloratum. The partially purified extract of V. coloratum (PPE-SVC) potently inhibited formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-induced superoxide anion generation and elastase release in a concentration-dependent manner with IC(50) values of 0.58+/-0.03 and 4.93+/-0.54 microg/ml, respectively. Furthermore, a new chalcone derivative, viscolin (4',4'-dihydroxy-2',3',6',3'-tetramethoxy-1,3-diphenylpropane), was isolated from PPE-SVC. Viscolin was demonstrated to inhibit superoxide anion generation and elastase release, as well as to accelerate resequestration of cytosolic calcium in FMLP-activated human neutrophils. Furthermore, the inhibitory effects of viscolin were reversed by protein kinase A (PKA) inhibitor, suggesting that PKA mediates the viscolin-caused inhibitions. Viscolin induced a substantial increase in cAMP levels, and that occurred through the inhibition of phosphodiesterase (PDE) activity but not an increase in adenylate cyclase function. Consistent with this, viscolin potentiated the PGE(1)-caused inhibition of superoxide anion release and calcium mobilization, as well as elevation of cAMP formation. These results demonstrate that inhibition of inflammatory responses in human neutrophils by viscolin is associated with an elevation of cellular cAMP through inhibition of PDE. Comparable results were also observed by PPE-SVC, indicating that the effect of PPE-SVC is at least partly mediated by viscolin. In summary, viscolin is a novel inhibitor of PDE and might be useful for treatment of neutrophilic inflammation.     

The structure of d(TpA), the major photoproduct of thymidylyl-(3'5')-deoxyadenosine.

Irradiation of the dinucleotide TpdA and TA-containing oligonucleotides and DNA produces the TA* photoproduct which was proposed to be the [2+2] cyclo-addition adduct between the C5-C6 double bonds of the T and the A [Bose,S.N., Kumar,S., Davies,R.J.H., Sethi,S.K. and McCloskey,J.A. (1984) Nucleic Acids Res. 12, 7929-7947]. The proposed structure was based on a variety of spectroscopic and chemical degradation studies, and the assignment of a trans-syn-I stereochemistry was based on an extensive 1H-NMR and molecular modeling study of the dinucleotide adduct [Koning,T.M.G., Davies,R.J.H. and Kaptein,R. (1990) Nucleic Acids Res. 18, 277-284]. However, a number of properties of TA* are not in accord with the originally proposed structure, and prompted a re-evaluation of the structure. To assign the 13C spectrum and establish the bond connectivities of the TA* photoproduct of TpdA [d(TpA)*], 1H-13C heteronuclear multiple-quantum coherence (HMQC) and heteronuclear multiple bond correlation (HMBC) spectra were obtained. The 13C shifts and connectivities were found to be inconsistent with the originally proposed cyclobutane ring fusion between the thymine and adenine, but could be explained by a subsequent ring-expansion reaction to give an eight-membered ring valence isomer. The new structure for the d(TpA)* resolves the inconsistencies with the originally proposed structure, and could have a stereochemistry that arises from the anti, anti glycosyl conformation found in B form DNA.     

Recombinant viral RdRps can initiate RNA synthesis from circular templates

The crystal structure of the recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) revealed extensive interactions between the fingers and the thumb subdomains, resulting in a closed conformation with an established template channel that should specifically accept single-stranded templates. We made circularized RNA templates and found that they were efficiently used by the HCV RdRp to synthesize product RNAs that are significantly longer than the template, suggesting that RdRp could exist in an open conformation prior to template binding. RNA synthesis using circular RNA templates had properties similar to those previously documented for linear RNA, including a need for higher GTP concentration for initiation, usage of GTP analogs, sensitivity to salt, and involvement of active-site residues for product formation. Some products were resistant to challenge with the template competitor heparin, indicating that the elongation complexes remain bound to template and are competent for RNA synthesis. Other products were not elongated in the presence of heparin, indicating that the elongation complex was terminated. Lastly, recombinant RdRps from two other flaviviruses and from the Pseudomonas phage phi6 also could use circular RNA templates for RNA-dependent RNA synthesis, although the phi6 RdRp could only use circular RNAs made from the 3'-terminal sequence of the phi6 genome.     

C-Terminal 23 kDa polypeptide of soybean Gly m Bd 28 K is a potential allergen

Gly m Bd 28 K is a major soybean (Glycine max Merr.) glycoprotein allergen. It was originally identified as a 28 kDa polypeptide in soybean seed flour. However, the full-length protein is encoded by an open reading frame (ORF) of 473 amino acids, and contains a 23 kDa C-terminal polypeptide of as yet unknown allergenic and structural characteristics. IgE-binding (allergenic potential) of the Gly m Bd 28 K protein including the 23 kDa C-terminal portion as well as shorter fragments derived from the full-length ORF were evaluated using sera from soy-sensitive adults. All of these sera contained IgE that efficiently recognized the C-terminal region. Epitope mapping demonstrated that a dominant linear C-terminal IgE binding epitope resides between residues S256 and A270. Alanine scanning of this dominant epitope indicated that five amino acids, Y260, D261, D262, K264 and D266, contribute most towards IgE-binding. A model based on the structure of the subunit of soybean -conglycinin revealed that Gly m Bd 28 K contains two cupin domains. The dominant epitope is on the edge of the first -sheet of the C-terminal cupin domain and is present on a potentially solvent-accessible loop connecting the two cupin domains. Thus, the C-terminal 23 kDa polypeptide of Gly m Bd 28 K present in soy products is allergenic and apparently contains at least one immunodominant epitope near the edge of a cupin domain. This knowledge could be helpful in the future breeding of hypoallergenic soybeans.Abbreviations Ara h 1 Arachis hypogaea allergen 1 - Ara h 3 Arachis hypogaea allergen 3 - BCA Bicinchoninic acid - Gly m Bd 28 K Glycine max band 28 kDa allergen - Gly m Bd 30 K Glycine max band 30 kDa allergen - Gly m Bd 68 K Glycine max band 68 kDa allergen - IgE Immunoglobulin E