Through the use of monospecific antibodies directed against hepatic cytochrome P-450j, an enzyme induced in rats treated with ethanol or isoniazid, we have purified from human liver the related cytochrome P-450 termed HLj. HLj resembles rat P-450j and P-450 LM3a, the homologous cytochrome in rabbit liver, in its NH2-terminal amino acid sequence, in being in highest concentration in liver microsome samples prepared from two patients intoxicated by ethanol and one patient given isoniazid, and in catalyzing the metabolic activation of the procarcinogen N-nitrosodimethylamine. Furthermore, each of nine human liver RNA samples contained a species of mRNA hybridizable to a cloned HLj cDNA. We conclude that HLj is related by structure, function, and some regulatory characteristics to rat P-450j and rabbit P-450 LM3a, cytochromes critical for metabolism of several clinically relevant cytotoxic and carcinogenic agents.

Polyclonal antibodies were produced in rabbits against purified cytochrome P-450j isolated from isoniazid-treated adult male rats. The monospecificity of immunoadsorbed antibody to cytochrome P-450j was demonstrated by Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays, and immunoblots. Immunoquantitation results indicated that rat liver microsomal cytochrome P-450j content decreases between 3 and 6 weeks of age in both the male and female animal. Several xenobiotics, such as Aroclor 1254, mirex, and 3-methylcholanthrene, repressed cytochrome P-450j levels when administered to male rats. Isoniazid, dimethyl sulfoxide, pyrazole, 4-methylpyrazole, and ethanol were inducers of cytochrome P-450j in rat liver although these compounds showed different inducing potencies. Microsomes from adult male rats with chemically induced diabetes also contained elevated levels of cytochrome P-450j compared to untreated animals. Cytochrome P-450j levels were measurable in kidney, whereas this isozyme was barely detectable in lung, ovaries, and testes; however, extrahepatic cytochrome P-450j was inducible by isoniazid. Approximately 80-90% of microsomal N-nitrosodimethylamine demethylation was inhibited by antibody to cytochrome P-450j whether the microsomes were isolated from untreated rats or animals administered inducers or repressors of cytochrome P-450j. The residual catalytic activity resistant to antibody inhibition may be a reflection of the inaccessibility of a certain amount of cytochrome P-450j due to interference by NADPH-cytochrome P-450 reductase based on results obtained with the reconstituted system. There was a good correlation (r2 = 0.87) between cytochrome P-450j content and N-nitrosodimethylamine demethylase activity in microsomes from rats of different ages and treated with various xenobiotics. The evidence presented indicates that cytochrome P-450j is the primary, and perhaps sole, microsomal catalyst of N-nitrosodimethylamine demethylation at substrate concentrations relevant to hepatocarcinogenesis induced by N-nitrosodimethylamine.

No abstract

We have isolated an allele of the essential DNA repair and transcription gene RAD3 that relaxes the restriction against recombination between short DNA sequences in Saccharomyces cerevisiae. Double-strand break repair and gene replacement events requiring recombination between short identical or mismatched sequences were stimulated in the rad3-G595R mutant cells. We also observed an increase in the physical stability of double-strand breaks in the rad3-G595R mutant cells. These results suggest that the RAD3 gene suppresses recombination involving short homologous sequences by promoting the degradation of the ends of broken DNA molecules.All organisms must repair the damage to their DNA that results from environmental stress and normal metabolism. Genetic recombination is one of several pathways that have evolved to repair this damage (13). This mode of repair can lead to deleterious consequences, however, because recombination between dispersed duplicate (ectopic) sequences can result in genome rearrangements or gene inactivation or both (42). These events are also implicated in human disease (20,24). Both DNA sequence length (2,26,51,53,58) and identity (6,18,36,43,45,47) are important determinants of the rate of recombination between repeated DNA sequences. What remains largely unclear is how short sequence length and mismatching hinder recombination. We took a genetic approach to studying the control of ectopic recombination in Saccharomyces cerevisiae. In a search for mutants that stimulate this recombination, we isolated an allele of the RAD3 gene (rad3-G595R) that increased the rate of recombination between sequences that share short lengths of perfect or imperfect homology.The RAD3 gene encodes a helicase (61) that is an essential component of the transcription and DNA repair complex TFIIH (factor B [10,66]) and is highly homologous to the human nucleotide excision repair gene XPD (60, 67). Many rad3 mutants have been isolated (13). These mutants exhibit a wide array of overlapping phenotypes including altered transcription (16), DNA repair (40,48,72), and recombination (37). One set of mutants, originally designated rem, were identified on the basis of their mutator and hyperrecombination phenotypes (14). The rem genes were subsequently found to be alleles of RAD3 and are thought to lead to the creation of recombinogenic double-strand breaks (37). Another mutant allele, rad3-2, is not hyperrecombinant but does decrease gene conversion tract length during intrachromosomal recombination (1). These diverse recombination phenotypes may be due to defects in the transcription of important recombination genes or to direct effects of mutant Rad3 proteins on recombination.White and Haber (71) analyzed the repair of double-strand breaks in DNA by homologous recombination between unlinked, duplicate sequences at the molecular level and showed that double-strand breaks are subject to extensive 5Ј-end degradation in wild-type cells. This processing is thought to be crucial for producing a single-stranded 3Ј end (3, 7...

Mutations in several nucleotide excision repair genes were found to affect the efficiency of recombination between short DNA sequences in Saccharomyces cerevisiae. These effects could be due to observed changes in the processing of recombination intermediates.Yeast (Saccharomyces cerevisiae) strains carrying mutant ura3 genes were transformed with DNA fragments containing the wild-type URA3 gene flanked by 36 to 866 bp of DNA homologous to the yeast HIS3 gene (Fig. 1). Only two types of Ura ϩ transformants were obtained: those resulting from DNA fragment integration into the HIS3 locus and those in which genomic ura3 sequences were restored to the wild type by either integration or gene conversion. We did not find URA3 sequences associated with any other locus (data not shown). As expected, the percentage of transformants of each type was dependent on the length of the HIS3 sequences at the end of the DNA fragment ( Fig. 1 and 2). The longer the length of HIS3 DNA, the higher the percentage of integrations at the HIS3 locus; however, the efficiency of recovery of Ura ϩ recombinants did not substantially change (Fig. 1). As in previous studies (3,11,13,20), we found that shorter HIS3 sequences resulted in fewer insertions into the HIS3 locus per unit length than longer sequences ( Fig. 1 and 2). This suggests that there is a mechanism for the suppression of recombination between sequences that have short lengths of homology.FIG. 1. Assay of recombination between DNA fragments and the genome. All substrates contained the URA3 gene (no shading) on a 1.2-kb DNA fragment. The first substrate consisted of the URA3 gene alone. The next five substrates consisted of the URA3 gene flanked by HIS3 DNA (shaded black; lengths in base pairs). The last three substrates contained 10, 20, and 30 bp, respectively, of heterologous (not homologous to the yeast genome) DNA (stippled) on both sides of the shortest HIS3 flanks. All strains were isogenic and contained wild-type (Wt) HIS3 and mutant ura3-1 alleles. The rad3-G595R (3) and rad1::LEU2 (23) strains were previously described. The rad3-20, rad2::TRP1, and rad10::LEU2 alleles were introduced by transformation. The transformation efficiency with the URA3-containing fragments (U ϩ ) was determined by dividing the number of Ura ϩ transformants obtained per microgram of DNA by the number of viable cells that were plated. The ranges from three independent determinations were reported. All Ura ϩ transformants were scored by replica plating for growth in the absence of histidine.