Degradation of structurally modified DNAs by bleomycin group antibiotics

Abstract

Bleomycin-mediated DNA strand scission has been shown to be diminished at certain sequences in proximity to 5-methylcytidines. We have investigated the molecular basis of this observed diminution using selective bleomycin (BLM) modifications at the C-terminus. Of the four different bleomycin congeners investigated, only bleomycin A2 and bleomycin BAPP were substantially affected by cytidine methylation. We have also examined the effect of other DNA modifications on bleomycin-mediated strand scission. Methylation at the N6 position of adenosine resulted in diminution of DNA cleavage by all four bleomycin congeners. The presence of bulky 5-(glucosyloxy)methyl groups in the major groove of T4 DNA had little effect on the efficiency of DNA strand scission mediated by bleomycin A2 or B2, suggesting the absence of important steric interactions between Fe(II).BLM and DNA in the major groove. In contrast, DNA cleavage mediated by bleomycin congeners was very sensitive to a major DNA conformational change, the B----Z transition. Salt and MgCl2 titrations of the DNA copolymers poly(dG-dC).poly(dG-dC) and poly(dG-MedC).poly(dG-MedC) demonstrated that bleomycin A2 and B2 did not cleave Z-DNA efficiently. In addition, circular dichroism titrations of these copolymers revealed that both bleomycin congeners increased the cation concentration necessary to induce the B----Z transition, implying that bleomycin preferentially binds to and stabilizes B-form DNA. These results are consistent with a model in which cytidine methylation at appropriate sequences of DNA is sufficient to induce subtle conformational changes that render the helix unreceptive to cleavage by some bleomycin congeners.

Site-specific cleavage of RNA by Fe(II).bleomycin.

“…Clearly, the dramatic difference in susceptibilities of these two tRNA precursors to degradation by Fe(II)-BLM argues for selective recognition of the tRNAHiS precursor at the level of tertiary structure; in fact, an alternate conformation for tRNATYr precursor has been suggested (20). This would parallel the alteration of DNA sequence selectivity as DNA conformation was altered (13,14,21).…”

“…Treatment with Fe(II)-BLM at a BLM to RNA nucleotide ratio similar to that used for tRNA His precursor effected cleavage at four sites, CCU-AACATA. subset of all 5'-GC-3' and 5'-GT-3' sites, although it has become increasingly clear that BLM is also responsive to DNA conformational alterations (13,14,19,21,23). Recognition of B-form DNA by Fe(II)-BLM primarily involves recognition of two adjacent bases and is optimal for 2 of the 10 possible distinguishable duplex sequences for a 2-base binding site (24).…”

“…Magliozzo et al (12) have reported degradation of yeast tRNAPhe by high concentrations of Fe(II)-BLM; product bands corresponding to <5-10% of the starting tRNA were observed by methylene blue staining of a polyacrylamide gel. Thin layer chromatographic analysis indicated products that comigrated with adenine and uracil.Although none ofthe foregoing studies suggested that RNA might constitute an efficient substrate for BLM, ongoing studies in this laboratory of BLM-mediated DNA oligonucleotide degradation have demonstrated that alteration of DNA conformation can significantly change the pattern and extent of cleavage normally obtained with B-form DNA (13,14). On the chance that the limited RNA cleavage observed (vide supra) might actually have resulted from highly efficient cleavage at a few conformationally unique sites, we studied several RNAs as substrates.…”

“…Although none ofthe foregoing studies suggested that RNA might constitute an efficient substrate for BLM, ongoing studies in this laboratory of BLM-mediated DNA oligonucleotide degradation have demonstrated that alteration of DNA conformation can significantly change the pattern and extent of cleavage normally obtained with B-form DNA (13,14). On the chance that the limited RNA cleavage observed (vide supra) might actually have resulted from highly efficient cleavage at a few conformationally unique sites, we studied several RNAs as substrates.…”

Genome Instability and Bleomycin Sensitivity Test

“…Stvaranje lomova u molekuli DNA pod utjecajem bleomicina smanjuje se u blizini 5-metilcitidina, a taj se utjecaj može primijetiti čak i 14 parova baza dalje od takvog mjesta (76,77).…”