The Mechanism of Dna Repair by Uracil-Dna Glycosylase: Studies Using Nucleotide Analogues

Abstract

2′,4′-Dideoxy-4′-methyleneuridine incorporated into oligodeoxynucleotides forms regular B-DNA duplexes as shown by T_m and CD measurements. Such oligomers are not cleaved by the DNA repair enzyme, UDG, which cleaves the glycosylic bond in dU but not in dT nor in dC nucleosides in single stranded and double stranded DNA. Differential binding of oligomers containing carbadU, 4′-thiodU, and dU residues to wild type and mutant UDG proteins identify an essential role for the furanose 4′-oxygen in recognition and cleavage of dU residues in DNA.     

The structural basis for substrate recognition by mammalian polynucleotide kinase 3' phosphatase

Mammalian polynucleotide kinase 3' phosphatase (PNK) plays a key role in the repair of DNA damage, functioning as part of both the nonhomologous end-joining (NHEJ) and base excision repair (BER) pathways. Through its two catalytic activities, PNK ensures that DNA termini are compatible with extension and ligation by either removing 3'-phosphates from, or by phosphorylating 5'-hydroxyl groups on, the ribose sugar of the DNA backbone. We have now determined crystal structures of murine PNK with DNA molecules bound to both of its active sites. The structure of ssDNA engaged with the 3'-phosphatase domain suggests a mechanism of substrate interaction that assists DNA end seeking. The structure of dsDNA bound to the 5'-kinase domain reveals a mechanism of DNA bending that facilitates recognition of DNA ends in the context of single-strand and double-strand breaks and suggests a close functional cooperation in substrate recognition between the kinase and phosphatase active sites.     

Expression of functional TRPA1 receptor on human lung fibroblast and epithelial cells

The transient receptor potential subfamily A member 1 (TRPA1) is a non-selective cation channel implicated in the pathogenesis of several airway diseases like asthma and chronic obstructive pulmonary disease (COPD). Most of the research on TRPA1 focuses on its expression and function in neuronal context; studies investigating non-neuronal expression of TRPA1 are lacking. In the present study, we show functional expression of TRPA1 in human lung fibroblast cells (CCD19-Lu) and human pulmonary alveolar epithelial cell line (A549). We demonstrate TRPA1 expression at both mRNA and protein levels in these cell types. TRPA1 selective agonists like allyl isothiocyanate (AITC), 4-hydroxynonenal (4-HNE), crotonaldehyde and zinc, induced a concentration-dependent increase in Ca+2 influx in CCD19-Lu and A549 cells. AITC-induced Ca+2 influx was inhibited by Ruthenium red (RR), a TRP channel pore blocker, and by GRC 17536, a TRPA1 specific antagonist. Furthermore, we also provide evidence that activation of the TRPA1 receptor by TRPA1 selective agonists promotes release of the chemokine IL-8 in CCD19-Lu and A549 cells. The IL-8 release in response to TRPA1 agonists was attenuated by TRPA1 selective antagonists. In conclusion, we demonstrate here for the first time that TRPA1 is functionally expressed in cultured human lung fibroblast cells (CCD19-Lu) and human alveolar epithelial cell line (A549) and may have a potential role in modulating release of this important chemokine in inflamed airways.     

Tumor necrosis factor-α and Muc2 mucin play major roles in disease onset and progression in dextran sodium sulphate-induced colitis

The sequential events and the inflammatory mediators that characterize disease onset and progression of ulcerative colitis (UC) are not well known. In this study, we evaluated the early pathologic events in the pathogenesis of colonic ulcers in rats treated with dextran sodium sulfate (DSS). Following a lag phase, day 5 of DSS treatment was found clinically most critical as disease activity index (DAI) exhibited an exponential rise with severe weight loss and rectal bleeding. Surprisingly, on days 1-2, colonic TNF-α expression (70-80-fold) and tissue protein (50-fold) were increased, whereas IL-1β only increased on days 7-9 (60-90-fold). Days 3-6 of DSS treatment were characterized by a prominent down regulation in the expression of regulatory cytokines (40-fold for IL-10 and TGFβ) and mucin genes (15-18 fold for Muc2 and Muc3) concomitant with depletion of goblet cell and adherent mucin. Remarkably, treatment with TNF-α neutralizing antibody markedly altered DSS injury with reduced DAI, restoration of the adherent and goblet cell mucin and IL-1β and mucin gene expression. We conclude that early onset colitis is dependent on TNF-α that preceded depletion of adherent and goblet cell mucin prior to epithelial cell damage and these biomarkers can be used as therapeutic targets for UC.     

Differential and Site Specific Impact of B Cells in the Protective Immune Response to Mycobacterium tuberculosis in the Mouse

Cell-mediated immune responses are known to be critical for control of mycobacterial infections whereas the role of B cells and humoral immunity is unclear. B cells can modulate immune responses by secretion of immunoglobulin, production of cytokines and antigen-presentation. To define the impact of B cells in the absence of secreted immunoglobulin, we analyzed the progression of Mycobacterium tuberculosis (Mtb) infection in mice that have B cells but which lack secretory immunoglobulin (AID^−/−µS^−/−mice). AID^−/−µS^−/− mice accumulated a population of activated B cells in the lungs when infected and were more susceptible to aerosol Mtb when compared to wild type (C57BL/6) mice or indeed mice that totally lack B cells. The enhanced susceptibility of AID^−/−µS^−/− mice was not associated with defective T cell activation or expression of a type 1 immune response. While delivery of normal serum to AID^−/−µS^−/− mice did not reverse susceptibility, susceptibility in the spleen was dependent upon the presence of B cells and susceptibility in the lungs of AID^−/−µS^−/−mice was associated with elevated expression of the cytokines IL-6, GM-CSF, IL-10 and molecules made by alternatively activated macrophages. Blocking of IL-10 signaling resulted in reversal of susceptibility in the spleens and lungs of AID^−/−µS^−/− mice. These data support the hypothesis that B cells can modulate immunity to Mtb in an organ specific manner via the modulation of cytokine production and macrophage activation.     

The crystal structure of yeast CCT reveals intrinsic asymmetry of eukaryotic cytosolic chaperonins

The cytosolic chaperonin CCT is a 1‐MDa protein‐folding machine essential for eukaryotic life. The CCT interactome shows involvement in folding and assembly of a small range of proteins linked to essential cellular processes such as cytoskeleton assembly and cell‐cycle regulation. CCT has a classic chaperonin architecture, with two heterogeneous 8‐membered rings stacked back‐to‐back, enclosing a folding cavity. However, the mechanism by which CCT assists folding is distinct from other chaperonins, with no hydrophobic wall lining a potential Anfinsen cage, and a sequential rather than concerted ATP hydrolysis mechanism. We have solved the crystal structure of yeast CCT in complex with actin at 3.8 Å resolution, revealing the subunit organisation and the location of discrete patches of co‐evolving ‘signature residues’ that mediate specific interactions between CCT and its substrates. The intrinsic asymmetry is revealed by the structural individuality of the CCT subunits, which display unique configurations, substrate binding properties, ATP‐binding heterogeneity and subunit–subunit interactions. The location of the evolutionarily conserved N‐terminus of Cct5 on the outside of the barrel, confirmed by mutational studies, is unique to eukaryotic cytosolic chaperonins.     

p185, an immunodominant epitope, is an autoantigen mimotope

An immunodominant peptide (p185(378-394)) derived from the c-erbB2 gene product, was recognized by an anti-DNA antibody, B3, and importantly by two classical DNA-binding proteins, Tgo polymerase and Pa-UDG. These reactivities were inhibited by DNA, confirming that the peptide mimicked DNA. BALB/c mice immunized with p185(378-394) developed significant titers of IgG anti-dsDNA antibodies. Screening of 39 human lupus sera revealed that 5% of these sera possessed reactivity toward p185(378-394). Representative mouse and human sera with anti-p185(378-394) reactivity bound intact p185, and this binding was inhibited by dsDNA. This is the first demonstration of a naturally occurring autoantigen mimotope. The present study identifies a potential antigenic stimulus that might trigger systemic lupus erythematosus in a subset of patients.     

Myeloid differentiation factor-2 interacts with Lyn kinase and is tyrosine phosphorylated following lipopolysaccharide-induced activation of the TLR4 signaling pathway

Stimulation with LPS induces tyrosine phosphorylation of numerous proteins involved in the TLR signaling pathway. In this study, we demonstrated that myeloid differentiation factor-2 (MD-2) is also tyrosine phosphorylated following LPS stimulation. LPS-induced tyrosine phosphorylation of MD-2 is specific; it is blocked by the tyrosine kinase inhibitor, herbimycin A, as well as by an inhibitor of endocytosis, cytochalasin D, suggesting that MD-2 phosphorylation occurs during trafficking of MD-2 and not on the cell surface. Furthermore, we identified two possible phospho-accepting tyrosine residues at positions 22 and 131. Mutant proteins in which these tyrosines were changed to phenylalanine had reduced phosphorylation and significantly diminished ability to activate NF-κB in response to LPS. In addition, MD-2 coprecipitated and colocalized with Lyn kinase, most likely in the endoplasmic reticulum. A Lyn-binding peptide inhibitor abolished MD-2 tyrosine phosphorylation, suggesting that Lyn is a likely candidate to be the kinase required for MD-2 tyrosine phosphorylation. Our study demonstrated that tyrosine phosphorylation of MD-2 is important for signaling following exposure to LPS and underscores the importance of this event in mediating an efficient and prompt immune response.