We have cloned the human mutY gene (hMYH) from both genomic and cDNA libraries. The human gene contains 15 introns and is 7.1 kb long. The 16 exons encode a protein of 535 amino acids that displays 41% identity to the Escherichia coli protein, which provides an important function in the repair of oxidative damage to DNA and helps to prevent mutations from oxidative lesions. The human mutY gene maps on the short arm of chromosome 1, between p32.1 and p34.3.The study of bacterial and yeast repair genes and their involvement in preventing mutations has facilitated the understanding of mutagenesis and repair in humans. A stunning example of this is the recent demonstration that inherited susceptibility to hereditary nonpolyposis colon cancer (HNPCC) is due to the inheritance of a defective copy of one of the human homologs of the bacterial mismatch repair system (4,6,8,19). Defects in either the Escherichia coli mutH, -L, or -S or the uvrD gene lead to a nonfunctional mismatch repair system and an increased rate of spontaneous mutations, the "mutator" phenotype (16). In humans, a mutator effect, easily detectable by observing instability of microsatellite repeats (8, 9), is also seen to result from defects in the mismatch repair system. This has led to renewed interest in characterizing mutator bacteria that might define additional repair systems and in finding their counterparts in humans.We have described two mutator genes in E. coli, the mutY and the mutM genes (5, 17), which work together to prevent mutations from certain types of oxidative damage, dealing in particular with the oxidized guanine lesion 8-oxodG (13). Figure 1 (see also reference 14) summarizes the concerted action of the enzymes encoded by these two genes, both of which are glycosylases. The MutM protein removes 8-oxodG from the DNA, and the resulting a purinic site is repaired to restore the GC base pair. Some lesions are not repaired before replication, which results in a GC-to-TA transversion at the next round of replication because polymerases involved in DNA replication incorporate A across from 8-oxoG between 5-fold and 200-fold more frequently than they incorporate C (22). However, the MutY protein removes the A across from 8-oxodG and repair synthesis restores a C most of the time (since polymerases involved in repair insert C in preference to A across from 8-oxoG [22]), allowing the MutM protein another opportunity to repair the lesion. In accordance with this, mutators lacking either the MutM or the MutY protein have an increase specifically in the GC-to-TA transversion (5, 17), and cells lacking both enzymes have an enormous increase in this base substitution (13). A third protein, the product of the mutT gene, prevents the incorporation of 8-oxodGTP by hydrolyzing the oxidized triphosphate back to the monophosphate (11), preventing AT-to-CG transversions.The human homolog of the E. coli mutT gene has been cloned and sequenced (20). Here we describe the characterization of the human homolog of the mutY gene (hMYH). The gene is 7.1 kb l...

IL-18-binding protein (IL-18BP) is a natural IL-18 inhibitor. Human IL-18BP isoform a was produced as fusion construct with human IgG1 Fc and assessed for binding and neutralizing IL-18. IL-18BP-Fc binds human, mouse, and rat IL-18 with high affinity (KD 0.3–5 nM) in a BIAcore-based assay. In vitro, IL-18BP-Fc blocks IL-18 (100 ng/ml)-induced IFN-γ production by KG1 cells (EC50 = 0.3 μg/ml). In mice challenged with an LD90 of LPS (15 mg/kg), IL-18BP-Fc (5 mg/kg) administered 10 min before LPS blocks IFN-γ production and protects against lethality. IL-18BP-Fc administered 10 min before LPS blocks IFN-γ production induced by LPS (5 mg/kg) with ED50 of 0.005 mg/kg. Furthermore, IL-18BP-Fc (5 mg/kg) abrogates LPS (5 mg/kg)-induced IFN-γ production even when administered 6 days before LPS but shows no effect when administered 9 or 12 days before LPS. Given 10 min before LPS challenge to mice primed 12 days in advance with heat-killed Propionibacterium acnes, IL-18BP-Fc prevents LPS-induced liver damage and IFN-γ and Fas ligand expression. Given at the moment of priming with P. acnes, IL-18BP-Fc decreases P. acnes-induced granuloma formation, macrophage-inflammatory protein-1α and macrophage-inflammatory protein-2 production and prevents sensitization to LPS. IL-18BP-Fc also prevents Con A-induced liver damage and IFN-γ and Fas ligand expression as well as liver damage induced by Pseudomonas aeruginosa exotoxin A or by anti-Fas agonistic Ab. In conclusion, IL-18BP can be engineered and produced in recombinant form to generate an IL-18 inhibitor, IL-18BP-Fc, endowed with remarkable in vitro and in vivo properties of binding and neutralizing IL-18.

Proteins containing the Nudix box "GX 5 EX 7 REUXE-EXGU" (where U is usually Leu, Val, or Ile) are Nudix hydrolases, which catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives. Here we report cloning and characterization of a human cDNA encoding a novel nudix hydrolase NUDT5 for the hydrolysis of ADP-sugars. The deduced amino acid sequence of NUDT5 contains 219 amino acids, including a conserved Nudix box sequence. The recombinant NUDT5 was expressed in Escherichia coli and purified to near homogeneity. At the optimal pH of 7, the purified recombinant NUDT5 catalyzed hydrolysis of two major substrates ADP-ribose and ADP-mannose with K m values of 32 and 83 M, respectively; the V max for ADP-mannose was about 1.5 times that with ADP-ribose. The murine NUDT5 homolog was also cloned and characterized. mNudT5 has 81% amino acid identity to NUDT5 with catalytic activities similar to NUDT5 under the optimal pH of 9. Both NUDT5 and mNudT5 transcripts were ubiquitously expressed in tissues analyzed with preferential abundance in liver. The genomic structures of both NUDT5 and mNudT5 were determined and located on human chromosome 10 and mouse chromosome 2, respectively. The role of NUDT5 in maintaining levels of free ADP-ribose in cells is discussed.

We have previously described the mutator alleles mutA and mutC, which map

We previously described Escherichia coli mutator tRNAs that insert glycine in place of aspartic acid and postulated that the elevated mutation rate results from generating a mutator polymerase. We suggested that the proofreading subunit of polymerase III, ɛ, is a likely target for the aspartic acid-to-glycine change that leads to a lowered fidelity of replication, since the altered ɛ subunits resulting from this substitution (approximately 1% of the time) are sufficient to create a mutator effect, based on several observations of mutDalleles. In the present work, we extended the study of specificmutD alleles and constructed 16 altered mutDgenes by replacing each aspartic acid codon, in series, with a glycine codon in the dnaQ gene that encodes ɛ. We show that three of these genes confer a strong mutator effect. We have also looked for new mutator tRNAs and have found one: a glycine tRNA that inserts glycine at histidine codons. We then replaced each of the seven histidine codons in the mutD gene with glycine codons and found that in two cases, a strong mutator phenotype results. These findings are consistent with the ɛ subunit playing a major role in the mutator effect of misreading tRNAs.