Transcription-coupled repair of 8-oxoguanine: requirement for XPG, TFIIH, and CSB and implications for Cockayne syndrome.

Analysis of transcription-coupled repair (TCR) of oxidative lesions here reveals strand-specific removal of 8-oxo-guanine (8-oxoG) and thymine glycol both in normal human cells and xeroderma pigmentosum (XP) cells defective in nucleotide excision repair. In contrast, Cockayne syndrome (CS) cells including CS-B, XP-B/CS, XP-D/CS, and XP-G/CS not only lack TCR but cannot remove 8-oxoG in a transcribed sequence, despite its proficient repair when not transcribed. The XP-G/CS defect uniquely slows lesion removal in nontranscribed sequences. Defective TCR leads to a mutation frequency at 8-oxoG of 30%-40% compared to the normal 1%-4%. Surprisingly, unrepaired 8-oxoG blocks transcription by RNA polymerase II. These data imply that TCR is required for polymerase release to allow repair and that CS results from defects in TCR of oxidative lesions.

Unidirectional heterologous receptor desensitization between both the fMLP and C5a receptor and the IL-8 receptor.

During inflammation neutrophils receive multiple signals that are integrated, allowing a single modified response. One mechanism for this discrimination is receptor desensitization, a process whereby ligand-receptor binding is disassociated from cell activation. We examined the effect of heterologous receptor desensitization on neutrophil chemotaxis, calcium mobilization, and arachidonic acid production, using interleukin-8 (IL-8), C5a, and N-formyl-methionyl-leucyl-phenylalanine (fMLP). We observed reciprocal inhibition with respect to chemotaxis. We demonstrated that homologous desensitization, with respect to the mobilization of intracellular calcium stores, lasted approximately 15 min. Heterologous desensitization between the fMLP receptor and the C5a receptor was reciprocal; either stimulant would diminish the cells' response to stimulation by the other for approximately 3-5 min. However, we observed a unidirectional heterologous desensitization of the IL-8 receptor by both the fMLP and the C5a receptor. This unidirectional heterologous desensitization was observed with respect to both calcium mobilization and arachidonic acid production (i.e., prestimulation of the IL-8 receptor had no effect on subsequent stimulation by either fMLP or C5a).

Sequences of complementary DNAs that encode the NA1 and NA2 forms of Fc receptor III on human neutrophils.

Two polymorphic forms of Fc receptor III (FcR III) are expressed on human neutrophils. These differ with respect to their apparent molecular masses after digestion with N-glycanase, and with respect to their reactivity with MAb Gran 11 and alloantisera which recognize determinants (NA1 and NA2) of the biallelic neutrophil antigen (NA) system. To determine the molecular basis for this polymorphism we isolated RNA from neutrophils of NA1NA1 and NA2NA2 homozygotes and synthesized corresponding cDNAs. cDNAs encoding FcR III were then amplified using the polymerase chain reaction, cloned, and sequenced. The cDNA that encodes FcR III on NA1NA1 neutrophils differed from the cDNA that encodes FcR III on NA2NA2 neutrophils at five nucleotides, predicting four amino acid substitutions. As a result, NA1 FcR III has only four potential N-linked glycosylation sites as compared with six in NA2 FcR III. The amino acid substitutions and differences in the number of potential N-linked glycosylation sites probably account for the different forms of neutrophil FcR III observed after digestion with N-glycanase and for the antigenic heterogeneity of this receptor.

Characterization of polymorphic forms of Fc receptor III on human neutrophils.

We characterized Fc receptor III (FcR III) on human neutrophils and found it to be heavily glycosylated and polymorphic. In some individuals, FcR III that had been digested with N-glycanase appeared after SDS-PAGE under reducing conditions as two bands with apparent molecular masses of 33 and 29 kD. In other individuals, N-glycanase-treated FcR III appeared as a single band with an Mr of either 33 or 29 kD. After SDS-PAGE of N-glycanase-treated FcR III under nonreducing conditions, the apparent Mr of each structural type was decreased, suggesting the presence of intramolecular disulfide bonds. Digestion of the 33-kD band and the 29-kD band with Staphylococcus aureus V8 protease yielded similar, but not identical, peptide maps. Thus, at least two polymorphic forms of FcR III are expressed on human neutrophils. The structural polymorphism of neutrophil FcR III correlated with previously described antigenic polymorphisms detected by monoclonal antibody Gran 11 and by alloantisera which recognize epitopes of the biallelic, neutrophil antigen (NA) system. Individuals whose neutrophils expressed the two-band structural type of FcR III were NA1NA2 heterozygotes. Individuals whose neutrophils expressed the single 33-kD band structural type were NA2NA2 homozygotes, and individuals whose neutrophils expressed the single 29-kD band structural type were NA1NA1 homozygotes. These findings indicate that antigenic and structural polymorphisms of human neutrophil FcR III are related and can be accounted for by differences at the level of primary protein structure.