Ultra-high resolution array painting facilitates breakpoint sequencing.

OBJECTIVE: To describe a considerably advanced method of array painting, which allows the rapid, ultra-high resolution mapping of translocation breakpoints such that rearrangement junction fragments can be amplified directly and sequenced. METHOD: Ultra-high resolution array painting involves the hybridisation of probes generated by the amplification of small numbers of flow-sorted derivative chromosomes to oligonucleotide arrays designed to tile breakpoint regions at extremely high resolution. RESULTS AND DISCUSSION: How ultra-high resolution array painting of four balanced translocation cases rapidly and efficiently maps breakpoints to a point where junction fragments can be amplified easily and sequenced is demonstrated. With this new development, breakpoints can be mapped using just two array experiments: the first using whole-genome array painting to tiling resolution large insert clone arrays, the second using ultra-high-resolution oligonucleotide arrays targeted to the breakpoint regions. In this way, breakpoints can be mapped and then sequenced in a few weeks.

The Cockayne Syndrome group B gene product is involved in general genome base excision repair of 8-hydroxyguanine in DNA.

Cockayne Syndrome (CS) is a human genetic disorder with two complementation groups, CS-A and CS-B. The CSB gene product is involved in transcription-coupled repair of DNA damage but may participate in other pathways of DNA metabolism. The present study investigated the role of different conserved helicase motifs of CSB in base excision repair. Stably transformed human cell lines with site-directed CSB mutations in different motifs within its putative helicase domain were established. We find that CSB null and helicase motif V and VI mutants had greater sensitivity than wild type cells to gamma-radiation. Whole cell extracts from CSB null and motif V/VI mutants had lower activity of 8-hydroxyguanine incision in DNA than wild type cells. Also, 8-hydroxyguanine accumulated more in CSB null and motif VI mutant cells than in wild type cells after exposure to gamma-radiation. We conclude that a deficiency in general genome base excision repair of selective modified DNA base(s) might contribute to CS pathogenesis. Furthermore, whereas the disruption of helicase motifs V or VI results in a CSB phenotype, mutations in other helicase motifs do not cause this effect. The biological functions of CSB in different DNA repair pathways may be mediated by distinct functional motifs of the protein.

Molecular characterization of an acidic region deletion mutant of Cockayne syndrome group B protein.

Cockayne syndrome (CS) is a human genetic disorder characterized by post-natal growth failure, neurological abnormalities and premature aging. CS cells exhibit high sensitivity to UV light, delayed RNA synthesis recovery after UV irradiation and defective transcription-coupled repair (TCR). Two genetic complementation groups of CS have been identified, designated CS-A and CS-B. The CSB gene encodes a helicase domain and a highly acidic region N-terminal to the helicase domain. This study describes the genetic characterization of a CSB mutant allele encoding a full deletion of the acidic region. We have tested its ability to complement the sensitivity of UV61, the hamster homolog of human CS-B cells, to UV and the genotoxic agent N-acetoxy-2-acetylaminofluorene (NA-AAF). Deleting 39 consecutive amino acids, of which approximately 60% are negatively charged, did not impact on the ability of the protein to complement the sensitive phenotype of UV61 cells to either UV or NA-AAF. Our data indicate that the highly acidic region of CSB is not essential for the TCR and general genome repair pathways of UV- and NA-AAF-induced DNA lesions.

The ATPase domain but not the acidic region of Cockayne syndrome group B gene product is essential for DNA repair.

Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA and CSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, the CSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways.

In vitro reactions of butadiene monoxide with single- and double-stranded DNA: characterization and quantitation of several purine and pyrimidine adducts.

We have previously shown that butadiene monoxide (BM), the primary metabolite of 1,3-butadiene, reacted with nucleosides to form alkylation products that exhibited different rates of formation and different stabilities under in vitro physiological conditions. In the present study, BM was reacted with single-stranded (ss) and double-stranded (ds) calf thymus DNA and the alkylation products were characterized after enzymatic hydrolysis of the DNA. The primary products were regioisomeric N-7-guanine adducts. N-3-(2-hydroxy-3-buten-1-yl)adenine and N-3-(1-hydroxy-3-buten-2-yl)adenine, which were depurinated from the DNA more rapidly than the N-7-guanine adducts, were also formed. In addition, N6-(2-hydroxy-3-buten-1-yl)deoxyadenosine and N6-(1-hydroxy-3-buten-2-yl)deoxyadenosine were detected and evidence was obtained that these adducts were formed by Dimroth rearrangement of the corresponding N-1-deoxyadenosine adducts, not while in the DNA, but following the release of the N-1-alkylated nucleosides by enzymatic hydrolysis. N-3-(2-hydroxy-3-buten-1-yl)deoxyuridine adducts, which were apparently formed subsequent to deamination reactions of the corresponding deoxycytidine adducts, were also detected and were stable in the DNA. Adduct formation was linearly dependent upon BM concentration (10-1000 mM), with adduct ratios being similar at the various BM concentrations. At a high BM concentration (750 mM), the adducts were formed in a linear fashion for up to 8 h in both ssDNA and dsDNA. However, the rates of formation of the N-3-deoxyuridine and N6-deoxyadenosine adducts increased 10- to 20-fold in ssDNA versus dsDNA, whereas the N-7-guanine adducts increased only slightly, presumably due to differences in hydrogen bonding in ssDNA versus dsDNA. These results may contribute to a better understanding of the molecular mechanisms of mutagenesis and carcinogenesis of both BM and its parent compound, 1,3-butadiene.

Characterization of four N-3-thymidine adducts formed in vitro by the reaction of thymidine and butadiene monoxide.

Four products were characterized from the reaction of thymidine with butadiene monoxide (BM), a known human mutagen and possible human carcinogen. These products were purified by HPLC and characterized as diastereomeric pairs of N-3-(1-hydroxy-3-buten-2-yl)thymidine and N-3-(2-hydroxy-3-buten-1-yl)thymidine based upon their UV spectra, 1H NMR and fast atom bombardment mass spectra. Incubation of thymidine with an excess of BM at pH 7.4 and 37 degrees C allowed calculation of the pseudo-first-order kinetic rate constants for the adduct formation, but when these rate constants were compared with the rates we previously determined with guanosine, adenosine and deoxycytidine, the results suggested a lower reactivity with thymidine in comparison with the other nucleosides. When incubations were carried out at lower BM concentrations, the formation of adducts appeared to be linearly dependent on BM concentration. The four thymidine adducts were completely stable for 1 week when incubated at 37 degrees C in pH 7.4 phosphate buffer. These results suggest that the interactions of BM with thymidine may play a role in the molecular mechanisms of mutagenesis and carcinogenesis of BM.

Chemical modification of deoxycytidine at different sites yields adducts of different stabilities: characterization of N3- and O2-deoxycytidine and N3-deoxyuridine adducts of butadiene monoxide.

Eight adducts were characterized from the reaction of deoxycytidine with the chemical carcinogen, butadiene monoxide (BM). They were identified as diastereomeric pairs of N3-(2-hydroxy-3-buten-1-yl)deoxycytidine, N3-(2-hydroxy-3-buten-1-yl)deoxyuridine, N3-(1-hydroxy-3-buten-2-yl)deoxyuridine, and O2-(2-hydroxy-3-buten-1-yl)deoxycytidine based on UV spectra, 1H NMR, FAB/MS, and stability studies. The N3-(2-hydroxy-3-buten-1-yl)deoxycytidine adducts were unstable at pH 7.4, 37 degrees C, and deaminated to the corresponding N3-deoxyuridine adducts with half-lives of 2.3 and 2.5 h. The N3-(1-hydroxy-3-buten-2-yl)deoxycytidine diastereomers were not detected, apparently because of faster rates of deamination compared to the N3-(2-hydroxy-3-buten-1-yl)deoxycytidine adducts. The corresponding four N3-deoxyuridine adducts were stable for up to 168 h. The O2-deoxycytidine adducts were unstable and decomposed with a half-life of 11 h. The N3-(2-hydroxy-3-buten-1-yl)deoxycytidine adducts were initially the major adducts formed upon reaction of deoxycytidine with BM at 37 degrees C in phosphate buffer (pH 7.4), but the corresponding N3-deoxyuridine adducts showed a lag in formation due to the time needed for deamination. The N3-(1-hydroxy-3-buten-2-yl)deoxyuridine and O2-deoxycytidine adducts had linear formation rates, but were formed to a lesser extent. Heating the reaction mixture at 80 degrees C for 1 h converted all N3-deoxycytidine adducts to the stable N3-deoxyuridine adducts. Incubation of deoxycytidine with an excess of BM at pH 7.4, 37 degrees C, followed by the extraction and heating steps allowed calculation of the pseudo-first-order kinetic rate constants for the four uridine adducts. If the heating step was eliminated, then the pseudo-first-order kinetic rate constants could be calculated for the N3-(2-hydroxy-3-buten-1-yl)deoxycytidine and O2-(2-hydroxy-3-buten-1-yl)deoxycytidine adducts. The rate constants for N3-(2-hydroxy-3-buten1-yl)deoxycytidine and the corresponding N3-(2-hydroxy-3-buten-1-yl)deoxyuridine were five- to sixfold the rate constants for the N3-(1-hydroxy-3-buten-2-yl)deoxyuridine and O2-(2-hydroxy-3-buten-1-yl)deoxycytidine adducts. Thus, the results show that the reaction of deoxycytidine with BM yields adducts at different sites with different rates of formation and stabilities. Understanding the chemical interactions of deoxycytidine with BM and the stability of the various adducts may contribute to a better understanding of the molecular mechanisms of mutagenesis and carcinogenesis of BM and the development of useful biomarkers of exposure.

Biochemistry of 1,3-butadiene metabolism and its relevance to 1,3-butadiene-induced carcinogenicity.

Recently, the roles of specific P450 isoforms, myeloperoxidase (MPO), GSH-S-transferase and epoxide hydrolase in the metabolism of 1,3-butadiene, and its major oxidative metabolite, butadiene monoxide (BM), were investigated. The results provided evidence for P450s 2A6 and 2E1 being major catalysts of 1,3-butadiene oxidation in human liver microsomes. cDNA-expressed human P450s 2E1, 2A6, and 2C9 catalyzed BM oxidation to meso- and (+/-)-diepoxybutane (DEB), but the rates of BM oxidation in mouse, rat, or human liver microsomes were much lower than the rates of 1,3-butadiene oxidation in these tissues. Human MPO catalyzed 1,3-butadiene oxidation to BM, but MPO incubations with BM did not yield DEB. Rates of BM formation in mouse and human liver microsomes were similar and were nearly 3.4-fold higher than that obtained with rat liver microsomes. However, rat liver epoxide hydrolase activity was nearly 2-fold higher than that of mouse liver microsomes. Rat and mouse liver GSH-S-transferases exhibited similar BM conjugation kinetics, but rats excreted more BM-mercapturic acids compared to mice given low equimolar doses of BM. BM reacted with guanosine and adenosine to yield N7-, N2-, and N1-guanosinyl and N6-adenosinyl adducts, respectively. These results may contribute to a better understanding of the biochemical basis of 1,3-butadiene-induced carcinogenicity.

Characterization of N1- and N6-adenosine adducts and N1-inosine adducts formed by the reaction of butadiene monoxide with adenosine: evidence for the N1-adenosine adducts as major initial products.

1,3-Butadiene is a known human mutagen and possible human carcinogen; however, the molecular mechanisms of its activity are poorly understood. We have previously shown that the primary metabolite, butadiene monoxide (BM), reacts with guanosine to form N1-, N2-, and N7-guanosine adducts. In this study we characterize the reaction of BM with adenosine; ten adducts identified as diastereomeric pairs of N1-(1-hydroxy-3-buten-2-yl)adenosine,N1-(2-hydroxy-3-buten-1 -yl)adenosine, N6-(1-hydroxy-3-buten-2-yl)adenosine,N6-(2-hydroxy-3-buten-+ ++1yl)adenosine, and N1-(1-hydroxy-3-buten-2-yl)inosine are characterized. The N6-adenosine and N1-inosine adducts were characterized by their UV spectra, 1H NMR, FAB/MS, and stability studies. The N6-adenosine and N1-inosine adducts were stable for up to 168 h at 37 degrees C in phosphate buffer (pH 7.4). The N1-adenosine adducts, which were unstable at pH 7.4 at 37 degrees C (half-life of 7 and 9.5 h for the two regioisomers), were characterized by their UV spectra and their ability to undergo the Dimroth rearrangement to yield the corresponding N6-adenosine adducts, or undergo deamination to yield the corresponding N1-inosine adducts. Upon the reaction of BM with adenosine in phosphate buffer (pH 7.4) at 37 degrees C, the N1-adenosine adducts were the first to be detected, with the N6-adenosine and N1-inosine adducts. showing a lag in formation possibly due to the time needed for rearrangement/deamination. Reaction of adenosine with an excess of BM in phosphate buffer (pH 7.4) at 37 degrees C, followed by extraction of the reaction mixture with ethyl ether to remove excess unreacted BM and incubation at 80 degrees C for 1 h, resulted in complete conversion of N1-adenosine adducts to the corresponding N6-adenosine and N1-inosine adducts. Under these conditions, adduct formation exhibited pseudo-first-order kinetics, with the combined N6-adenosine adducts being formed 3-fold more favorably than the combined N1-inosine adducts. When incubations were carried out at lower BM concentrations, the N6-adenosine adducts remained the major detectable adducts at all concentrations. These results show that adenosine, in addition to guanosine, can lead to multiple adducts when incubated with BM, and may be useful in development of biomarkers for exposure to 1,3-butadiene. Characterization of the N1-adenosine adducts and their rearrangement/deamination products may also contribute to the understanding of mutagenic and carcinogenic mechanisms of 1,3-butadiene.

Synthesis and biochemical characterization of N1-, N2-, and N7-guanosine adducts of butadiene monoxide.

1,3-Butadiene is a known rodent carcinogen, but the molecular mechanisms of its carcinogenicity are poorly understood. Butadiene monoxide (BM), a known mutagenic metabolite of 1,3-butadiene, was previously shown to react with guanosine to yield two N7-guanine adducts. In the present study, eight guanosine adducts of BM were purified and characterized as diastereomeric pairs of N7-(2-hydroxy-3-buten-1-yl)guanosine (G-1 and G-3), N7-(1-hydroxy-3-buten-2-yl)guanosine (G-2 and G-5), N2-(1-hydroxy-3-buten-2-yl)guanosine (G-4 and G-7), and N1-(1-hydroxy-3-buten-2-yl)guanosine (G-6 and G-8) on the basis of stability studies and analyses by UV, 1H NMR, and fast atom bombardment mass spectrometry. While the N7-adducts exhibited half-lives of approximately 50 (G-1 and G-3) and 90 h (G-2 and G-5) upon incubation for 192 h in 100 mM phosphate buffer (pH 7.4) at 37 degrees C, the N1- and N2-adducts remained stable. When guanosine was reacted with excess BM in phosphate buffer (pH 7.4) at 37 degrees C, adduct formation exhibited pseudo-first-order kinetics, with the N7-adducts being formed approximately 10-fold more favorably than the N1- and N2-adducts. When incubations were carried out at lower BM concentrations, the N7-adducts remained the major detectable adducts, but the N2-adducts were also detectable at equimolar BM and guanosine concentrations, and the N1-adducts were detectable at a 5-fold molar excess of BM. These results, which provide clear evidence that guanosine can be alkylated at multiple sites following 1,3-butadiene exposure, may aid in the development of useful biomarkers for exposure to 1,3-butadiene. The results may also contribute to a better understanding of the molecular mechanisms of 1,3-butadiene-induced carcinogenicity.