Formation of C1' located sugar radicals from X-irradiated cytosine nucleosides and -tides in BeF2 glasses and frozen aqueous solutions.

Free radical formation in nucleosides and nucleotides containing cytosine as base was studied after X-irradiation at 77 K of samples prepared in frozen aqueous BeF2 glasses and in frozen aqueous solutions by means of electron spin resonance (ESR) spectroscopy. By comparison with radical patterns from the cytosine base and from 1-CH3-cytosine, by using specifically base-deuterated nucleosides and by comparison between the 2'-deoxy- and the ribonucleotide it could be demonstrated that a radical at the C1'-position of the sugar was formed in nucleosides and nucleotides in both matrices. Quantitative analysis showed that in the BeF2 glass an initial population of about 10% of substrate located species due to this radical was present at 77 K and developed to about 25% after decay of the .OH (.OD) radicals at about 140 K. This was taken as proof that at least part of these radicals were formed from .OH radicals. In frozen aqueous solutions about 20% of C1' located sugar radicals were found to be present at 77 K, the population remaining roughly constant with increasing temperature to 140 K. The mechanistic findings of these unexpected results are discussed in terms of mobile .OH radicals and/or hole transfer in the glass and in the glassy regions of the frozen aqueous solutions.

[Molecular nature of the mutations in gene ADE2 in Saccharomyces cerevisiae yeasts. III. Determination of the correlation of the GC AT pairs in genes ADE1 and ADE2]. / Izuchenie molekuliarnoi prirody mutatsii v gene ADE2 u drozhzhei Saccharomyces cerevisiae. Soobshchenie III. Opredelenie sootnosheniia par GTs i AT v genakh ADE1 i ADE2.

Lethal and mutagenic effects and the nature of mutations induced by decay of 32P incorporated into yeast cell DNA as 32P-deoxyguanosine monophosphate (32PdGMP) and 32P-thymidine monophosphate (32P-TMP), were studied. The lethal efficiency per 32P decay is independent of a labelled nucleotide incorporated into DNA. However, the mutagenic efficiency in ADE1, ADE2 genes per 32P decay is approximately 3 times greater for 32PdGMP than for 32P-TMP. This suggests that ADE1, ADE2 genes contain about 3 times more GC base pairs than AT pairs. Variations in a relative frequencies of GC leads to AT and AT leads to GC transitions were obtained depending upon a nucleotide labelled.

Photochemical cross-linking of neighboring residues in protein-nucleic acid complexes: rnase and pyrimidine nucleotide inhibitors.

Iradiation of the stable complexes formed between RNase and its competitive inhibitors cytidine 2'(3'),5'-diphosphate (pCp), and uridine 2'(3'),5'-diphosphate (pUp), resulted in covalent bond formation between the pyrimidine nucleotides and the enzyme. The photochemical reactions were initiated by ultraviolet light of lambda greater than 300 mn, employing acetone as a photosensitizer. This method was found to yield less undesired by-products, particularly photolyzed amino acids and aggregates resulting from protein-to-protein cross-linking, than the direct method of irradiation with light of lambda 260 nm. Tryptic digrestion of the modified protein, and chromatographic analysis of the peptides thus obtained, revealed a single and specific peptide which bacame covalently linked to both nucleotide inhibitors. The amino acid composition of this peptide is consistent with the sequence Asn-67-Arg-85 of RNase. Part of this peptide (residues 78 through 83) is close to the enzyme's binding site for the pyrimidine moiety of the nucleotides. Denatured RNase failed to cross-link to the inhibitors, and the extent of pUp cross-linking could be reduced by the addition of another competitive inhibitor (3'-UMP). Finally, the presence of excess inhibitor in the irradiation mixture did not lead to nonspecific cross-linking. This indicates that specificity is also achieved due to the fact that unbound excited inhibitor molecules do not react with the protein but prefer to follow different and faster reaction paths.