IL-18 and IL-33 elicit Th2 cytokines from basophils via a MyD88- and p38alpha-dependent pathway.

IL-4 and IL-13 are instrumental in the development and progression of allergy and atopic disease. Basophils represent a key source of these cytokines and produce IL-4 and IL-13 when stimulated with IL-18, a member of the IL-1 family of cytokines. Comparative analyses of the effects of caspase-1-dependent IL-1 family cytokines on basophil IL-4 and IL-13 production have not been performed, and the signaling pathway proteins required for FcepsilonRI-independent Th2 cytokine production from basophils remain incompletely defined. Using mouse bone marrow-derived cultured basophils, we found that IL-4 and IL-13 are produced in response to IL-18 or IL-33 stimulation. IL-18- or IL-33-mediated Th2 cytokine production is dependent on MyD88 and p38alpha signaling proteins. In addition, basophil survival increased in the presence of IL-18 or IL-33 as a result of increased Akt activation. Studies in vivo confirmed the potency of IL-18 and IL-33 in activating cytokine release from mouse basophils.

Replication of an oxidized abasic site in Escherichia coli by a dNTP-stabilized misalignment mechanism that reads upstream and downstream nucleotides.

Abasic sites (AP) and oxidized abasic lesions are often referred to as noninstructive lesions because they cannot participate in Watson-Crick base pairing. The aptness of the term noninstructive for describing AP site replication has been called into question by recent investigations in E. coli using single-stranded shuttle vectors. These studies revealed that the replication of templates containing AP sites or the oxidized abasic lesions resulting from C1'- (L) and C4'-oxidation (C4-AP) are distinct from one another, suggesting that structural features other than Watson-Crick hydrogen bonds contribute to controlling replication. The first description of the replication of the abasic site resulting from formal C2'-oxidation (C2-AP) is presented here. Full-length and single-nucleotide deletion products are observed when templates containing C2-AP are replicated in E. coli. Single nucleotide deletion formation is largely dependent upon the concerted effort of pol II and pol IV, whereas pol V suppresses frameshift product formation. Pol V utilizes the A-rule when bypassing C2-AP. In contrast, pol II and pol IV utilize a dNTP-stabilized misalignment mechanism to read the upstream and downstream nucleotides when bypassing C2-AP. This is the first example in which the identity of the 3'-adjacent nucleotide is read during the replication of a DNA lesion. The results raise further questions as to whether abasic lesions are noninstructive lesions. We suggest that abasic site bypass is affected by the local biopolymer structure in addition to the structure of the lesion.

In vitro replication and repair of DNA containing a C2'-oxidized abasic site.

Abasic lesions are unable to form Watson-Crick hydrogen bonds with nucleotides. Nonetheless, polymerase and repair enzymes distinguish between various oxidized abasic lesions, as well as from nonoxidized abasic sites (AP). The C2-AP lesion is produced when DNA is exposed to gamma-radiolysis. Its effects on polymerases and repair enzymes are unknown. A recently reported method for the chemical synthesis of oligonucleotides containing C2-AP at a defined site was utilized for studying the activity of Klenow exo(-) and repair enzymes on templates containing the lesion. The C2-AP lesion has a similar effect on Klenow exo(-) as do AP and C4-AP sites. Deoxyadenosine is preferentially incorporated opposite C2-AP, but extension of the primer past the lesion is strongly blocked. C2-AP is incised less efficiently by exonuclease III and endonuclease IV than are other abasic lesions. Furthermore, although a Schiff base between C2-AP and endonuclease III can be chemically trapped, the location of the 3'-phosphate alpha with respect to the aldehyde prevents beta-elimination associated with the lyase activity of type I base excision repair enzymes. The interactions of the C2'-oxidized abasic site with Klenow exo(-) and repair enzymes suggest that the lesion will be mutagenic and that it will be removed by strand displacement synthesis and flap endonuclease processing via a long patch repair mechanism.

A comprehensive comparison of DNA replication past 2-deoxyribose and its tetrahydrofuran analog in Escherichia coli.

Apurinic/apyrimidinic (AP) sites are alkali labile lesions that, when encountered during DNA replication, can block polymerases or potentially result in mutagenic events. Owing to the instability of 2-deoxyribose lesions (AP), a chemically stable tetrahydrofuran analog (F) is often used as a model of abasic sites. A comparison of the two lesions in Saccharomyces cerevisiae revealed that the model lesion and 2-deoxyribose have distinct in vivo effects. Comprehensive comparative analyses of F and AP have not been carried out in Escherichia coli. We conducted a side-by-side investigation of F and AP in E.coli to compare their biological effects and interactions with SOS polymerases. Both lesions were examined in SOS-induced and uninduced cells. Our studies reveal that in uninduced E.coli the effects of individual polymerases in the replication of plasmids containing F or AP are distinct. However, when cells are SOS-induced, the biological effects of F and AP are similar.

Effects of the C4'-oxidized abasic site on replication in Escherichia coli. An unusually large deletion is induced by a small lesion.

The C4'-oxidized abasic site (C4-AP) is produced in DNA as a result of oxidative stress by a variety of agents. For instance, the lesion accounts for approximately 40% of the DNA damage produced by the antitumor antibiotic bleomycin. The effect of C4-AP on DNA replication in Escherichia coli was determined using the restriction endonuclease and postlabeling (REAP) method. Three-nucleotide deletion products are the sole products observed following replication of plasmids containing C4-AP under SOS conditions in wild-type cells. Full-length products are formed in varying amounts depending upon the local sequence in wild-type cells under non-SOS-induced conditions. The "A-rule" is followed for the formation of substitution products. C4-AP is the first example of a DNA lesion that produces significant levels of three-nucleotide deletions in a variety of sequence contexts. Experiments carried out in cells lacking specific polymerases reveal that formation of three-nucleotide deletion products results from a coordinated effort involving pol II and pol IV. This is the first example in which these SOS inducible polymerases are shown to work in concert during lesion bypass. Three-nucleotide deletions are not observed during the replication of other abasic lesions, and are rarely produced by bulky adducts. The effect of C4-AP on DNA replication suggests a significant role for this lesion in the cytotoxicity of bleomycin. Formation of the C4-AP lesion may also be responsible for the formation of mutant proteins containing single-amino acid deletions that exhibit altered phenotypes.

Mutagenic effects of 2-deoxyribonolactone in Escherichia coli. An abasic lesion that disobeys the A-rule.

Abasic sites are often referred to as noninstructive lesions. The C1'-oxidized abasic site (2-deoxyribonolactone, L) is produced by several DNA damaging agents, including gamma-radiolysis and the neocarzinostatin chromophore (NCS). The effects of a C1'-oxidized abasic site incorporated at a defined site in single-stranded plasmid were examined in SOS polymerase-proficient and -deficient Escherichia coli. For comparison, experiments utilizing plasmids containing an abasic site (AP) were carried out side by side. In contrast to plasmid containing AP, dA and dG were incorporated most often when plasmid containing L was replicated. The ratio of dG:dA incorporation depended upon local sequence and varied from 0.9 to 2.2. High levels of translesion incorporation of dA are consistent with previous observations that treatment of DNA with the neocarzinostatin chromophore resulted in large amounts of G.C --> A.T transitions [Povirk and Goldberg (1986) Nucleic Acids Res. 14, 1417] and support the proposal that L is the source of these mutations. Both abasic lesions were 100% lethal in triple knockout cells lacking pol II, pol IV, and pol V. Analysis of translesion synthesis in repair-deficient cells revealed that pol V played a significant role in replication of L and AP. Significant levels of -1 frameshifts were formed in 5'-d(CL) sequences in the presence of pol V and were the exclusive product in pol V-deficient cells. Frameshift products were not formed when the nucleotide on the 5'-side of L was either dT or dG. Deleting pol II or pol IV had only modest effects on replication of L-containing plasmid but significantly decreased the amount of -1 frameshift product formed from an AP lesion. Experiments carried out side by side using otherwise identical plasmids containing an AP site illustrate the distinct properties of these two abasic lesions and that neither should be thought of as noninstructive.

In vitro effects of a C4'-oxidized abasic site on DNA polymerases.

Oxidative damage to DNA produces abasic sites resulting from the formal hydrolysis of the nucleotides' glycosidic bonds, along with a variety of oxidized abasic sites. The C4'-oxidized abasic site (C4-AP) is produced by several DNA-damaging agents. This lesion accounts for approximately 40% of the DNA damage produced by bleomycin. The effect of a C4'-oxidized abasic site incorporated at a defined site in a template was examined on Klenow fragments with and without 3' --> 5' exonuclease activity. Both enzymes preferentially incorporated dA > dG >> dC, T opposite C4-AP. Neither enzyme is able to extend the primer past the lesion. Experiments with regular AP sites in an otherwise identical template indicate that Klenow does not differentiate between these two disparate abasic sites. Extension of the primer by alternative polymerases pol II, pol II exo(-), pol IV, and pol V was examined. Pol II exo(-) was most efficient. Qualitative translesion synthesis experiments showed that pol II exo(-) preferentially incorporates T opposite C4-AP, followed in order by dG, dA, and dC. Thymidine incorporation opposite C4'-AP is distinct from the pol II exonuclease interaction with a regular AP site in an otherwise identical template. These in vitro experiments suggest that bypass polymerases may play a crucial role in survival of cells in which C4-AP is produced, and unlike a typical AP site, the C4-AP lesion may not follow the "A-rule". The interaction between bypass polymerases and a C4-AP lesion could explain the high levels of G:C --> T:A transversions in cells treated with bleomycin.

Cross-linking of 2-deoxyribonolactone and its beta-elimination product by base excision repair enzymes.

2-Deoxyribonolactone (3) is produced in DNA as a result of reaction with a variety of DNA damaging agents. The lesion undergoes beta-elimination to form a second metastable electrophilic product (4). In this study, DNA containing 2-deoxyribonolactone (3) and its beta-elimination product (4) are generated at specific sites using a photolabile nucleotide precursor. 2-Deoxyribonolactone is not incised by any of the 8 AP lyases tested. One enzyme, Escherichia coli endonuclease III, cross-links to 3, and the lesion strongly inhibits excision of typical abasic sites by this enzyme. Two of the enzymes, FPG and NEIL1 known to cleave normal abasic sites (1) by effecting beta,delta-elimination form cross-links to the butenolide lesion (4). The observed results are ascribable to characteristics of the enzymes and the lesions. These enzymes are also important for the removal of oxidative base lesions. These results suggest that high concentrations of 3 and 4 may exert significant effects on the repair of normal AP site and oxidative base lesions in cells by reducing the cellular activity of these BER enzymes either via cross-linking or competing with binding to the BER enzymes.