Structure of bleomycin-induced DNA double-strand breaks: predominance of blunt ends and single-base 5' extensions

Abstract

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)

The natural absence of RPA1N domain did not impair Leishmania amazonensis RPA-1 participation in DNA damage response and telomere protection

“…DNA damage induced by phleomycin elicited a G1/S cell cycle arrest in L. amazonensis promastigotes It is known that in Saccharomyces cerevisiae, 50 μg mL − 1 of phleomycin for 60-120 min induces a checkpoint response that is controlled by the ATMrelated TEL1 protein (Nakada et al 2003) and that in most eukaryotes DNA DSBs (Moore, 1988;Povirk et al 1989) are repaired by homologous recombination (HR) (Belenguer et al 1995;Sugiyama and Kowalczykowski, 2002;Stauffer and Chazin, 2004). Leishmania major promastigotes exposed to 10 μg mL − 1 of phleomycin for 24-48 h also showed a damage response with detectable levels of the RAD51, a protein that commonly participates in damage repair by HR.…”

Treatment of cancer with cryochemotherapy

“…Table 1 demonstrates that bleomycin has the ability to significantly increase cell damage at thermal conditions typical to those on the outer rim of a cryosurgical lesion. Bleomycin molecules form complexes with Fe 2+ and O 2 that directly generate breaks on DNA through the nucleophilic attack of the C' 4 of the desoxyribose, particularly at the GC sequences (Povirk et al, 1989;Mir et al, 1996). Since bleomycin must penetrate the cell to affect cell survival, the data presented in Table 1 suggests that the bleomycin has entered the cells during freezing and that it is affecting cells which otherwise would have survived the freezing protocol.…”

Activation of PARP-1 in response to bleomycin depends on the Ku antigen and protein phosphatase 5

“…By contrast, radiomimetic glycopeptides of the bleomycin family are free radical-based DNA damaging agents that produce much higher levels of DSBs with relatively uniform and simple DNA ends (Povirk, 1996). For example, about 10% the DNA damage generated by bleomcyin are DSBs with 5 0 phosphate and 3 0 phosphoglycolate termini with either blunt DNA ends or DNA ends offset by one basepair 5 0 overhang (Povirk et al, 1989).…”