The mechanism of free base formation from DNA by bleomycin. A proposal based on site specific tritium release from Poly(dA.dU)

Poly(dA.dU), which is specifically tritiated at the 1'-, 2'- (ribo configuration), 3'-, or 4'-position of deoxyuridine, has been synthesized and the fate of the tritium has been determined upon degradation of the polymer by bleomycin, Fe(II), and O2. No tritium is labilized from the 1'-3H-labeled polymer as 3H2O; however, the resulting 3-(uridin-1'-yl)-2-propenal (uracil propenal) has the expected specific activity. The 2'-3H-labeled polymer affords 3H2O and no label in the uracil propenal. This result and the lack of solvent incorporation into the uracil propenal suggest that proton abstraction from C-2' to afford the trans-propenal is highly stereospecific. For the 3'-3H-labeled polymer, 3H2O is formed and the specific activity of the uracil propenal is identical to that of the deoxyuridine. This suggests that the labilization of the 3'-H is exclusively associated with free uracil formation. 3H2O is also formed from the 4'-3H-labeled polymer. These findings along with previous studies are consistent with the formation of uracil propenal and free uracil by the trapping of the initially formed 4'-radical species by O2 or by a monooxygen species, respectively.     

DNA structure influences sequence specific cleavage by bleomycin.

We have examined the cleavage of several synthetic DNA sequences by iron(II)-bleomycin. We find that, although bleomycin cuts mixed sequence DNAs with a preference for GC = GT > GA >> GG, it efficiently cleaves regions of (AT)n cutting exclusively at ApT, not TpA. Isolated ApT steps show very little cleavage while blocks of three or more contiguous ATs are cut as efficiently as GpT. This cleavage is specific for (AT)n, since sequences of the type (TAA)n.(TTA)n and (ATT)n.(AAT)n are hardly cut at all. No cleavage is observed at ApC or CpA within sequences of the type (AC)n.(GT)n; regions of An.Tn are also not cut. Although the cobalt-bleomycin complex (which binds to but does not cleave DNA) yields good DNase I footprints at GT and GC sites, no footprints are observed within (AT)n, suggesting that although the cleavage reaction is efficient, the binding affinity is relatively weak. We propose a model in which bleomycin cleavage is determined by local DNA structure, while strong binding requires the presence of a guanine residue.     

Functional analogues of bleomycin: DNA cleavage by bleomycin and hemin-intercalators

New hemin-intercalators (Hem-G's) that cleave DNA were synthesized, on the basis of 2-amino-6-methyldipyrido[1,2-alpha:3',2'-d]imidazole (Glu-P-1) as an intercalator moiety. Hem-G's, which possess an intramolecular ligand of the ferrous ion (a histidine or imidazole moiety), cleave DNA very efficiently and act at guanine-pyrimidine sequences preferentially. Bleomycin (BLM) also cleaved DNA with the same base-sequence selectivity shown by Hem-G's. The 5'-terminus of the DNA fragments cleaved by Hem-G's or by BLM is a phosphoryl group, while the 3'-terminus of the cleaved DNA fragments does not possess a 3'-phosphoryl group. There are more than three kinds of 5'-end 32P-labeled DNA fragments, which can be substrates of terminal deoxynucleotidyl transferase (TdT). One of the 3'-termini of the cleaved DNA fragments is a 3'-hydroxy group. The mobility of the 3'-end 32P-labeled DNA fragment cleaved by Hem-G's or by BLM corresponds to the removal of pyrimidine bases having guanine at the 5'-side. The mobility of one kind of the cleaved 5'-end 32P-labeled DNA fragments corresponds to the removal of guanine having pyrimidine at the 3'-side, followed by 3'-dephosphorylation. We propose that there exist plural mechanisms for DNA cleavage by Hem-G's or by BLM. The deduced structures of the cleaved DNA fragments suggest that one of the mechanisms involves deletion of two nucleotide units from DNA.     

Inhibition of DNA polymerase alpha by bleomycin

Bleomycin is an important anti-tumor agent which works primarily through it's degradation of DNA template. Using synthetic single (poly[dA]-oligo-[dT]) and double stranded (poly[dA-dT]) templates, we noted significant inhibition when the BLM resistant homopolymer was used. Furthermore, when each of the components of the DNA polymerase assay were treated with bleomycin separately, followed by removal of bleomycin, significant inhibition (35%) of the enzyme was observed. The limited inhibition of DNA polymerase by BLM was attributed to residual activity of the enzyme-inhibitor complex.     

DNA double-strand breaks and alkali-labile bonds produced by bleomycin.

Both in linear T2 DNA, analyyzed by velocity sedimentation, and in supercoiled Col EL DNA, analyzed by gel electrophoresis, the number of double-strand breaks produced by bleomycin was directly propotional to the number of single-strand breaks and was far greater than the number expected from random coincidence of single-strand breaks, suggesting that the bleomycin-induced double-strand breaks occur as an independent event. In Col EL DNA, at least twice as many single-strand breaks were found under alkaline assay conditions as were found under neutral conditions, showing the production of alkaline-labile bonds by bleomycin.     

The kinetics of DNA damage by bleomycin in mammalian cells.

The kinetics of DNA damage by bleomycin (BLM) was assessed by measuring the amount of DNA breakage induced by BLM at different doses, treatment lengths, and treatment temperatures. DNA degradation was measured with the alkaline unwinding method. Comparison of the curves of DNA cleavage by BLM leads to the conclusion that low doses (1-5 micrograms/ml) and short treatments (5-15 min) produce marked damage in the DNA. High increases in BLM concentration produce relatively small increases in DNA damage above the levels obtained with low doses. Extension of treatment times does not increase the DNA degradation above the rate observed with 15-min treatments. The repair of DNA damage starts at about 15 min after the initiation of treatment. The mending of DNA breaks is very fast and extensive when BLM is no longer present. Repair not only implies the closing of DNA nicks, but very likely the degradation of the BLM molecules intercalated in the DNA interrupting the reactions responsible for the generation of free radicals. Persistence of BLM in the cell environment facilitates the replacement of degraded BLM molecules by new ones. This produces the persistent production of free radicals and the establishment of a balance between the processes of DNA damage and repair.     

Specificity of DNA cleavage by ultrasound

Cleavage of double-stranded DNA fragments with known nucleotide sequences upon sonication at 22 and 44 kHz was studied by PAGE. The cleavage rate was shown to depend on the fragment size, pH, ionic strength, and temperature. Double-strand breaks occurred preferentially in 5′-CpG-3′ dinucleotides. The strand was broken between C and G so that the phosphate group was at the 5′ side of G in the products. The cleavage rate proved to depend on the sequences flanking the cleavage site. The character of cleavage changed in the presence of Pt-bis-netropsin, a sequence-specific ligand that alters the local conformation of DNA.     

Neocarzinostatin-induced DNA base release accompanied by staggered oxidative cleavage of the complementary strand

Treatment of an end-labeled DNA restriction fragment with the nonprotein chromophore of neocarzinostatin induced lesions which, after treatment with endonuclease IV or putrescine, were expressed as site-specific double-strand breaks. Analysis of the termini at cleavage sites in each strand showed that the neocarzinostatin-induced lesions consisted of an apurinic/apyrimidinic site plus a closely opposed break in the complementary strand. The break always occurred opposite the base two positions upstream from the apurinic/apyrimidinic site and had the 3'-phosphate and 5'-aldehyde termini characteristic of neocarzinostatin-induced breaks. This positioning suggests that neocarzinostatin simultaneously attacks two DNA sugars on opposite edges of the minor groove. The sequence specificity for formation of apurinic/apyrimidinic sites with closely opposed breaks reflected that of neocarzinostatin-induced mutagenesis. The potent mutagenicity of these lesions may be attributable to the presence of closely opposed damage in both DNA strands.     

Iron . bleomycin . DNA system evidence of a long-lived bleomycin . iron . oxygen intermediate

When Fe(II) is added to a bleomycin. DNA mixture in the presence of air a long-lived $\text{(}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 45}\text{minutes}\text{)}$ EPR silent species (I′) is formed; the circular dichroism and absorption spectra of which have been characterized. This complex slowly decays yielding a ferric complex (III′) analogous to the well known low spin Fe(III). BLM species.     

Specificity of DNA binding and methylation by the M.FokI DNA methyltransferase

The M.FokI adenine-N(6) DNA methyltransferase recognizes the asymmetric DNA sequence GGATG/CATCC. It consists of two domains each containing all motifs characteristic for adenine-N(6) DNA methyltransferases. We have studied the specificity of DNA-methylation by both domains using 27 hemimethylated oligonucleotide substrates containing recognition sites which differ in one or two base pairs from GGATG or CATCC. The N-terminal domain of M.FokI interacts very specifically with GGATG-sequences, because only one of the altered sites is modified. In contrast, the C-terminal domain shows lower specificity. It prefers CATCC-sequences but only two of the 12 star sites (i.e. sites that differ in 1 bp from the recognition site) are not accepted and some star sites are modified with rates reduced only 2-3-fold. In addition, GGATGC- and CGATGC-sites are modified which differ at two positions from CATCC. DNA binding experiments show that the N-terminal domain preferentially binds to hemimethylated GGATG/C(m)ATCC sequences whereas the C-terminal domain binds to DNA with higher affinity but without specificity. Protein-protein interaction assays show that both domains of M.FokI are in contact with each other. However, several DNA-binding experiments demonstrate that DNA-binding of both domains is mutually exclusive in full-length M.FokI and both domains do not functionally influence each other. The implications of these results on the molecular evolution of type IIS restriction/modification systems are discussed.     

Strand scission of deoxyribonucleic acid by neocarzinostatin, auromomycin, and bleomycin: studies on base release and nucleotide sequence specificity

The nucleotide sequence specificity of neocarzinostatin (NCS), auromomycin (AUR), bleomycin (Blm), phleomycin (Phlm), and tallysomycin (Tlm) has been determined by using these antibiotics and their associated chromophores to create strand scissions in end-labeled restriction fragments of DNA and then determining the base sequence of the oligonucleotides formed. NCS and the NCS chromophore induce similar patterns of cleavage in DNA fragments labeled at the 5' terminus. The pattern produced by the AUR chromophore also resembles that of its holoantibiotic. Dithiothreitol enhances the rate of cleavage of DNA by the AUR chromophore but does not alter the sequence specificity. The results suggest that the polypeptide component of AUR and NCS serves primarily as a carrier for the chromophore. When tested with a fragment labeled at the 3' terminus, the products of NCS and AUR cleavage do not display the patterns of chemically produced oligonucleotides cleaved at phosphodiester bonds, suggesting that the 5' terminus is modified by a sugar fragment. NCS primarily attacks thymine (75% of the total bases attacked) and, to a lesser extent, adenine (19%) and cytosine (6%). AUR preferentially attacks guanine (67% of total bases), while attacking less often thymine (24%) and adenine (9%). Bleomycin and its analogues preferentially cleave purine--pyrimidine (5' leads to 3') and pyrimidine--pyrimidine (3' leads to 5') sequences. All (5' leads to 3') GT and GC sequences were cleaved. Phlm G and Phlm-Pep are less active than bleomycin toward purines while Tlm was more active. The patterns of cleavage produced by Blm A2 and Blm B6 are similar, while those produced by Phlm-Pep, Phlm G, Blm-B1', and Blm-Pep resemble one another. The cleavage pattern of Tlm shows quantitative differences from the other analogues tested. Differences between bleomycin and its analogues may be related to structural differences in these molecules.     

Characterization of chain breakage in DNA induced by bleomycin

Both deoxyribose moieties and phosphodiester bonds of DNA are destroyed by the action of bleomycin. A malonic aldehyde-like compound is produced in the drug-treated DNA. Phosphodiester bond rupture results in the production of 30 % of hydroxyl groups and 70 % of phosporyl groups in the 5′-termini of bleomycintreated DNA. Very few or perhaps none of the 3′-termini are phosphorylated.     

The sequence specificity of bleomycin damage in three cloned DNA sequences that differ by a small number of base substitutions

The DNA sequence specificity of the cancer chemotherapeutic agent, bleomycin, has been investigated in three clones of human alpha RI-DNA. The three 340-base pair alpha RI-DNA sequences were almost identical in their nucleotide sequence enabling the study of subtle effects of base substitutions on bleomycin cleavage. By utilizing densitometer scanning and statistical analysis of the degree of bleomycin DNA cleavage, we found 17 significant differences between the three DNA sequences. Eleven of these differences could be attributed to base substitutions close to the dinucleotide cleavage site. However, six of the differences were at positions two or more base pairs from the base substitution sites. The significant differences were up to 12 base pairs from base substitutions. It is proposed that these long range effects are due to base substitutions causing microvariation in the DNA structure to which bleomycin cleavage is sensitive.     

DNA base flipping by a base pair-mimic nucleoside

On the basis of non-covalent bond interactions in nucleic acids, we synthesized the deoxyadenosine derivatives tethering a phenyl group (X) and a naphthyl group (Z) by an amide linker, which mimic a Watson-Crick base pair. Circular dichroism spectra indicated that the duplexes containing X and Z formed a similar conformation regardless of the opposite nucleotide species (A, G, C, T and an abasic site analogue F), which was not observed for the natural duplexes. The values among the natural duplexes containing the A/A, A/G, A/C, A/T and A/F pairs differed by 5.2 kcal mol(-1) while that among the duplexes containing X or Z in place of the adenine differed by only 1.9 or 2.8 kcal mol(-1), respectively. Fluorescence quenching experiments confirmed that 2-amino purine opposite X adopted an unstacked conformation. The structural and thermodynamic analyses suggest that the aromatic hydrocarbon group of X and Z intercalates into a double helix, resulting in the opposite nucleotide base flipping into an unstacked position regardless of the nucleotide species. This observation implies that modifications at the aromatic hydrocarbon group and the amide linker may expand the application of the base pair-mimic nucleosides for molecular biology and biotechnology.     

DNA degradation by bleomycin: evidence for 2'R-proton abstraction and for C-O bond cleavage accompanying base propenal formation

Reaction of poly(dA-[2'S-3H]dU) with activated bleomycin yields [3H]uracil propenal that completely retains the tritium label. In contrast, we have previously shown that reaction of poly(dA-[2'R-3H]dU) with activated bleomycin affords unlabeled uracil propenal [Wu, J. C., Kozarich, J. W., & Stubbe, J. (1983) J. Biol. Chem. 258, 4694-4697]. We have also prepared both cis- and trans-thymine propenals by chemical synthesis and have observed that the trans isomer is the exclusive product of the bleomycin reaction. Moreover, the cis isomer was found to be stable to the conditions of bleomycin-induced DNA degradation. Taken together, these results establish that the formation of trans-uracil propenal occurs via an anti-elimination mechanism with the stereospecific abstraction of the 2'R proton. The question of phosphodiester bond cleavage during base propenal formation has also been addressed by the analysis of the fate of oxygen-18 in poly(dA-[3'-18O]dT) upon reaction with activated bleomycin. The 5'-monophosphate oligonucleotide ends produced from thymine propenal formation have been converted to inorganic phosphate by the action of alkaline phosphatase, and the phosphate has been analyzed for 18O content by 31P NMR spectroscopy. The oxygen-18 is retained in the inorganic phosphate, establishing that the formation of thymine propenal by activated bleomycin proceeds with C-O bond cleavage at the 3'-position.