2 research outputs found
DNA Methylation Reduces Binding and Cleavage by Bleomycin
In
a recent study, we described the enhanced double-strand cleavage
of hairpin DNAs by FeĀ·bleomycin (FeĀ·BLM) that accompanies
increasingly strong binding of this antitumor agent and suggested
that this effect may be relevant to the mechanism by which BLM mediates
its antitumor effects. Because the DNA in tumor cells is known to
be hypomethylated on cytidine relative to that in normal cells, it
seemed of interest to study the possible effects of methylation status
on BLM-induced double-strand DNA cleavage. Three hairpin DNAs found
to bind strongly to bleomycin, and their methylated counterparts,
were used to study the effect of methylation on bleomycin-induced
DNA degradation. Under conditions of limited DNA cleavage, there was
a significant overall decrease in the cleavage of methylated hairpin
DNAs. Cytidine methylation was found to result in decreased BLM-induced
cleavage at the site of methylation and to result in enhanced cleavage
at adjacent nonmethylated sites. For two of the three hairpin DNAs
studied, methylation was accompanied by a dramatic decrease in the
binding affinity for FeĀ·BLM, suggesting the likelihood of diminished
double-strand cleavage. The source of the persistent binding of BLM
by the third hairpin DNA was identified. Also identified was the probable
molecular mechanism for diminished binding and cleavage of the methylated
DNAs by BLM. The possible implications of these findings for the antitumor
selectivity of bleomycin are discussed
A Short DNA Sequence Confers Strong Bleomycin Binding to Hairpin DNAs
Bleomycins A<sub>5</sub> and B<sub>2</sub> were used to study the
structural features in hairpin DNAs conducive to strong BLMāDNA
interaction. Two members of a 10-hairpin DNA library previously found
to bind most tightly to these BLMs were subsequently noted to share
the sequence 5ā²-ACGC (complementary strand sequence 5ā²-GCGT).
Each underwent double-strand cleavage at five sites within, or near,
an eight base pair region of the DNA duplex which had been randomized
to create the original library. A new hairpin DNA library was selected
based on affinity for immobilized FeĀ(III)Ā·BLM A<sub>5</sub>.
Two of the 30 newly identified DNAs also contained the sequence 5ā²-ACGC/5ā²-GCGT.
These DNAs bound to the FeĀ(II)Ā·BLMs more tightly than any DNA
characterized previously. Surface plasmon resonance confirmed tight
FeĀ(III)Ā·BLM B<sub>2</sub> binding and gave an excellent fit for
a 1:1 binding model, implying the absence of significant secondary
binding sites. FeĀ(II)Ā·BLM A<sub>5</sub> was used to assess sites
of double-strand DNA cleavage. Both hairpin DNAs underwent double-strand
cleavage at five sites within or near the original randomized eight
base region. For DNA <b>12</b>, four of the five double-strand
cleavages involved independent single-strand cleavage reactions; DNA <b>13</b> underwent double-strand DNA cleavage by independent single-strand
cleavages at all five sites. DNA <b>14</b>, which bound FeĀ·BLM
poorly, was converted to a strong binder (DNA <b>15</b>) by
insertion of the sequence 5ā²-ACGC/5ā²-GCGT. These findings
reinforce the idea that tighter DNA binding by FeĀ·BLM leads to
increased double-strand cleavage by a novel mechanism and identify
a specific DNA motif conducive to strong BLM binding and cleavage