In order to examine the structure of bleomycin-induced DNA double-strand breaks, defined-sequence DNA was labeled in each strand at a single restriction site and treated with bleomycin. Various double-stranded fragments resulting from bleomycin-induced double-strand breaks were isolated, denatured, and run on sequencing gels to determine the sites of cleavage in each strand. For virtually every double-strand break, the cleavage site in one strand was a pyrimidine in a G-Py sequence, reflecting a specificity similar to that of bleomycin-induced single-strand cleavage. However, the cleavage site in the complementary strand was seldom a G-Py sequence, and was usually a site where single-strand cleavage was infrequent. When the sequence at the double-strand break was G-Py-Py', the break at Py was usually accompanied by a break at the base directly opposite Py, resulting in blunt ends. When the sequence was G-Py-Pu, the break at Py was usually accompanied by a break at the base opposite Pu, resulting in single-base 5' extensions. Double-strand breaks with 3' extensions, such as would result from cleavage of two C residues in a self-complementary G-C sequence, were conspicuously absent. These data provide further evidence that bleomycin-induced double-strand breaks do not result from coincidence of independent site-specific single-strand breaks.(ABSTRACT TRUNCATED AT 250 WORDS)

Using various end-labeled, defined-sequence DNA substrates, we examined bleomycin-induced damage at several G'C base pairs which correspond to mutational hot spots. The most frequent lesions detected were single-strand breaks and single apu inic/apyrimidinic (AP) sites at the C residue, suggesting that this was the primary site of damage. Strand breaks and AP sites also occurred, but less frequently, at a secondary damage site-i.e., the directly opposed G residue in the complementary strand. However, damage at the secondary site occurred only when a strand break was present at the primary site, and AP sites at the primary site were never accompanied by closely opposed damage in the complementary strand. Thus, formation of a strand break at the primary damage site was a necessary though not sufficient condition for attack at the secondary site. Similar patterns were seen at other sequences attacked by bleomycin, although primary and secondary sites were sometimes staggered by one nucleotide position rather than directly opposed. These and other results suggest a mechanism of double-strand cleavage in which bleomycin Is reactivated during formation of the first strand break, and the reactivated drug subsequently attacks the complementary strand at a specific position which is not normally a site of bleomycin-induced cleavage. Regeneration of activated bleomycin could result from a reaction between Fe(w)'bleomycin and a 4'-peroxyl derivative of deoxyribose, both produced during formation of the strand break.The fact that bleomycin produces double-strand breaks with single-hit kinetics suggests that these lesions result from a single interaction between bleomycin and DNA (1, 2). However, the chemistry of activated bleomycin (3,4) gives no suggestion of bifunctionality, and the mechanism of doublestrand cleavage has remained uncertain. We recently showed that most bleomycin-induced double-strand breaks consist of a "primary" site, which follows the normal G-Y (Y =pyrim-idine nucleoside) specificity characteristic of bleomycininduced single-strand cleavage, and a secondary site (in the opposite strand), which is seldom a G-X sequence and is usually a site where single-strand cleavage is rare (2). In addition to double-strand breaks, bleomycin induces formation of apurinic/apyrimidinic (AP) sites with closely opposed strand breaks, and these bivalent lesions have been implicated in bleomycin-induced mutagenesis in A phage (5, 6). To clarify the mechanism by which bleomycin effects concomitant damage to both DNA strands, we have examined the detailed structure of bivalent lesions induced by bleomycin at specific sites in defined-sequence DNA substrates, particularly those corresponding to mutational hot spots.MATERIALS AND METHODS Materials. Recombinant plasmids were constructed by cloning either a synthetic 25-mer (pc1245, Fig. 1 Analysis of DNA Cleavage. Nondenaturing and denaturing gel electrophoresis and elution of fragments from gels have been described (2). Except in experiments employing hydrazine, all e...

Mitochondrial DNA control region sequences were determined in 109 unrelated German Caucasoid individuals from north west Germany for both hypervariable regions 1 (HV1) and 2 (HV2) and 100 polymorphic nucleotide positions (nps) were found, 63 in HV1 and 37 in HV2. A total of 100 different mtDNA lineages was revealed, of which 7 were shared by 2 individuals and 1 by 3 individuals. The probability of drawing a HV1 sequence match within the north west Germans or within published sets of south Germans and west Austrians is similar (within a factor of 2) to drawing a sequence match between any two of these three population samples. Furthermore, HV1 sequences of 700 male inhabitants of one village in Lower Saxony were generated and these showed a nearly linear increase of the number of different haplotypes with increasing number of individuals, demonstrating that the commonly used haplotype diversity measure (Nei 1987) for population samples tends to underestimate mtDNA diversity in the actual population.

This study reports mtDNA polymorphisms in both hypervariable segments HV1 and HV2 of the non coding D-loop region from 60 unrelated Koreans. In contrast to two previous Korean data base studies, mtDNA was extracted separately from pulp tissue and root dentin of teeth obtained from dentists. Dentin turned out to be a reliable source of mitochondrial DNA. This can be of practical importance in forensic identification case work after a long post-mortem interval since pulp decomposes rapidly. The extraction method is explained in detail. The mtDNA polymorphisms obtained from 60 teeth of unrelated Koreans were compared with the already existing Korean data base.

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