Edith F. Yamasaki

Ubiquitin-Family Modifications of Topoisomerase I in Camptothecin-Treated Human Breast Cancer Cells

Camptothecins kill mammalian cells by stabilizing topoisomerase I-DNA strand passing intermediates that are converted to lethal double strand DNA breaks in DNA replication fork collisions. Camptothecin-stabilized topoisomerase I-DNA cleavage intermediates in mammalian cells are uniquely modified by ubiquitin-family proteins. The structure, composition, and function of these ubiquitin-family modifications are poorly understood. We have used capillary liquid chromatography-nanospray tandem mass spectrometry to analyze the endogenous ubiquitin-family modifications of topoisomerase I purified from camptothecin-stabilized topoisomerase I-DNA cleavage complexes in human breast cancer cells. Peptides shared by SUMO-2 and SUMO-3 were abundant, and a peptide unique to SUMO-2 was identified. Ubiquitin was also identified in these complexes. No SUMO-1 peptide was detected in human topoisomerase I-DNA cleavage complexes. Identical experiments with purified SUMO paralogues showed that SUMO-1 was well digested by our protocol and that fragments were easily analyzed by LC-MS/MS. Spiking experiments with purified SUMO paralogues determined that we could detect as little as 0.5 SUMO-1 residue per topoisomerase I molecule. These results indicate that SUMO-1 is below this detection level and that SUMO-2 or a mixture of SUMO-2 and SUMO-3 predominates. SUMO-1 capping seems unlikely to be limiting the growth of SUMO-2/3 chains formed on camptothecin-stabilized topoisomerase I-DNA cleavage complexes.

Mutational activation of H-ras oncogene transformability by alkylnitrosourea-induced DNA damage

To assess the role of DNA alkylation damage in oncogene activation, plasmid DNA containing H-ras proto-oncogene (p220-EC) and oncogene (p220-EJ) were treated with increasing concentrations of carcinogenic methylnitrosourea (MNU) and ethylnitrosourea (ENU). The modified plasmid DNA were analyzed by transfection-transformation of the NIH/3T3-recipient cells. Treatment with varying doses of MNU (0.1-5 mM) and ENU (1-15 mM) did not result in the inactivation of the plasmid containing target genes. A transformation efficiency of greater than 40% was observed upon treatment of H-ras oncogene with the highest doses of the alkylating agents. The morphologically transformed foci obtained with alkylated p220-EC ranged from 2.8 to 0.3/microgram MNU alkylated and 1.6 to 0.6/microgram ENU alkylated plasmid DNA. A significant proportion of the morphological transformants exhibited growth in soft agar. The HpaII/MspI restriction length polymorphism (RFLP) at codon 12 of H-ras exon-1 was detected with 4 independently isolated clones obtained from MNU-alkylated p220-EC transfections. Allele-specific in situ gel hybridization with a battery of codon 12 and codon 61 oligonucleotide probes confirmed these RFLPs to be due to sequence changes at codon 12. No clone with sequence changes in the H-ras codon 61 could be detected. The data indicate that a high degree of in vitro alkylation damage of the target gene is necessary to elicit mutational activation of H-ras in transfection-transformation assay. Low frequency notwithstanding, the data demonstrate that DNA alkylation damage at critical target sites can initiate neoplastic cellular transformation.

Quantitative analysis of carbodiimide modified dna and immunoprobing by adduct specific antibodies

Antibodies have been raised against N-cyclohexyl-N-(4-methylmorpholinium)ethyl carbodiimide (CMC) modified single-stranded DNA and characterized by competitive and non-competitive immunoassays to be highly specific for CMC base adduct in homopolymers poly(dG), poly(dT) and DNA. The antibodies recognize picogram concentrations of CMC treated DNA with no cross reactivity to at least 1000-fold excess of unmodified DNA or CMC treated poly(dA). The detection limit of antibodies at 1.4 fmol CMC adduct allows quantitation at a CMC/base ratio of 4.6.10(-7). Based upon single modified base-containing synthetic oligomers, a 7-fold higher binding preference is observed for CMC modified thymine than guanine bases. CMC binding to supercoiled DNA is found to depend upon reaction temperature and ionic strength. CMC-modified supercoiled SV40 and ColE1 DNA, exhibit specific antibody binding proportional to the DNA concentration and extent of CMC modification. However, antibody binding observed is independent of the conformation or strandedness of CMC-modified DNA. DNA extensively modified with CMC retains its inherent capacity to specifically and quantitatively hybridize with complementary DNA immobilized to membranes upon direct blotting or Southern transfers from gels. Hybridized CMC-DNA, through antibody binding, provides for the sensitive and non-isotopic detection of the target DNA sequences.

Chapter 4 Solid Phase Immunoassay for Determining the Inosine Content in Transfer RNA

Publisher Summary Determination of the inosine content in transfer RNA (tRNA) is an important consideration due to its potential role in altering gene expresxadsion. Inosine is found in tRNA in the first (wobble) position of the anticodon, and in that position, it can influence codon recognition and, thereby, protein synthesis. As a result, quantitation of changes in the inosine content of tRNA is of value for examining the role of this tRNA modification in translational regulation. Reversed-phase high-performance liquid chromatography (HPLC) offers one means for determining the inosine content in hydrolyzed tRNA. Nucleosides in the tRNA hydrolysate are resolved by the HPLC method, and under many circumstances, the inosine content can be established by simple on-line detection based on ultraviolet (UV) absorbance. However, it is difficult to separate inosine from some of the other modified nucleosides found in unfractionated tRNAs, so alternative detection methods are needed. Inosinexad specific, high titer and high affinity antibodies were raised in rabbits against inosine-keyhole limpet hemocyanin conjugate. An enzyme-linked immunosorbent assay (ELISA) was developed and inosine was quantitated by competitive inhibition with immobilized antigen. The immunological approach for the quantitation of inosine has proven most effective when the assay is coupled to the reversed-phase HPLC separation of nucleosides in tRNA hydrolysates, but other uses are also likely.