Dynamics of Bleomycin Interaction with a Strongly Bound Hairpin DNA Substrate, and Implications for Cleavage of the Bound DNA

Bozeman, Trevor C.; Nanjunda, Rupesh; Tang, Chenhong; Liu, Yang; Segerman, Zachary J.; Zaleski, Paul A.; Wilson, W. David; Hecht, Sidney M.

doi:10.1021/ja306233e

Cited by 19 publications

(38 citation statements)

References 45 publications

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“…13 Each of these DNAs was found to have a single strong binding site for Fe(III)·BLM, but one or more significantly weaker binding sites. In contrast, each was found to undergo cleavage at a number of sites, affording multiple single- and double-strand cleavage products (SI Figure S1 and Figure 9).…”

Section: Discussionmentioning

confidence: 99%

Hairpin DNA Sequences Bound Strongly by Bleomycin Exhibit Enhanced Double-Strand Cleavage

Roy

Hecht

2014

J. Am. Chem. Soc.

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Clinically used bleomycin A5 has been employed in a study of double-strand cleavage of a library of 10 hairpin DNAs originally selected on the basis of their strong binding to bleomycin. Each of the DNAs underwent double-strand cleavage at more than one site, and all of the cleavage sites were within, or in close proximity to, an eight-base-pair region of the duplex that had been randomized to create the original library. A total of 31 double-strand cleavage sites were identified on the 10 DNAs, and 14 of these sites were found to represent coupled cleavage sites, that is, events in which one of the two strands was always cleaved first, followed by the associated site on the opposite strand. Most of these coupled sites underwent cleavage by a mechanism described previously by the Povirk laboratory and afforded cleavage patterns entirely analogous to those reported. However, at least one coupled cleavage event was noted that did not conform to the pattern of those described previously. More surprisingly, 17 double-strand cleavages were found not to result from coupled double-strand cleavage, and we posit that these cleavages resulted from a new mechanism not previously described. Enhanced double-strand cleavages at these sites appear to be a consequence of the dynamic nature of the interaction of Fe·BLM A5 with the strongly bound hairpin DNAs.

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Section: Discussionmentioning

confidence: 99%

Hairpin DNA Sequences Bound Strongly by Bleomycin Exhibit Enhanced Double-Strand Cleavage

Roy

Hecht

2014

J. Am. Chem. Soc.

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“…35 Two important observations were made during this study. First, each of the hairpin DNAs studied, including 2, 4 and 5 , formed a single, strongly bound complex with Fe(III)•BLM but at least one, and likely multiple, more weakly bound and more transient complexes.…”

Section: Discussionmentioning

confidence: 90%

“…41-43 The presence of large numbers of closely spaced DNA cleavage sites on both strands of the hairpin DNA library suggests that numerous double-strand cleavage sites may be present. While the extent of double-strand cleavage of randomly chosen DNAs occurs at a level of frequency well beyond can be accounted for by the random accumulation of single strand breaks, 40,41 the hairpin DNAs studied here, which were selected for tight binding to BLM, have recently been shown to sequester Fe•BLM, 35 such that double-strand cleavage might plausibly result from two independent cleavage events more frequently than for randomly chosen DNAs.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Bleomycin-Mediated Cleavage of a Hairpin DNA Library

2013

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A study of BLM A5 was conducted using a previously isolated library of hairpin DNAs found to bind strongly to metal free BLM. The ability of Fe(II)•BLM to effect cleavage on both the 3' and 5'-arms of the hairpin DNAs was characterized. The strongly bound DNAs were found to be efficient substrates for Fe•BLM A5-mediated hairpin DNA cleavage. Surprisingly, the most prevalent site of BLM-mediated cleavage was found to be the 5′-AT-3′ dinucleotide sequence. This dinucleotide sequence, and other sequences generally not cleaved well by BLM when examined using arbitrarily chosen DNA substrates, were apparent when examining the library of ten hairpin DNAs. In total, 132 sites of DNA cleavage were produced by exposure of the hairpin DNA library to Fe•BLM A5. The existence of multiple sites of cleavage on both the 3′- and 5′-arms of the hairpin DNAs suggested that some of these might be double-strand cleavage events. Accordingly, an assay was developed with which to test the propensity of the hairpin DNAs to undergo double-strand DNA damage. One hairpin DNA was characterized using this method, and gave results consistent with earlier reports of double-strand DNA cleavage, but with a sequence selectivity different from those reported previously.

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“…The data are listed in Tables 2 and 3 , where they are also compared with the values determined previously for hairpin DNA 2 . 12 …”

Section: Resultsmentioning

confidence: 99%

“…Based on reproducibility of results, the errors in the strong binding constants and kinetics constants are ±15%. b Data from ref ( 12 ). …”

Section: Resultsmentioning

confidence: 99%

A Short DNA Sequence Confers Strong Bleomycin Binding to Hairpin DNAs

et al. 2014

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Bleomycins A5 and B2 were used to study the structural features in hairpin DNAs conducive to strong BLM–DNA 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 5′-ACGC (complementary strand sequence 5′-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 Fe(III)·BLM A5. Two of the 30 newly identified DNAs also contained the sequence 5′-ACGC/5′-GCGT. These DNAs bound to the Fe(II)·BLMs more tightly than any DNA characterized previously. Surface plasmon resonance confirmed tight Fe(III)·BLM B2 binding and gave an excellent fit for a 1:1 binding model, implying the absence of significant secondary binding sites. Fe(II)·BLM A5 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 12, four of the five double-strand cleavages involved independent single-strand cleavage reactions; DNA 13 underwent double-strand DNA cleavage by independent single-strand cleavages at all five sites. DNA 14, which bound Fe·BLM poorly, was converted to a strong binder (DNA 15) by insertion of the sequence 5′-ACGC/5′-GCGT. These findings reinforce the idea that tighter DNA binding by Fe·BLM leads to increased double-strand cleavage by a novel mechanism and identify a specific DNA motif conducive to strong BLM binding and cleavage.

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Dynamics of Bleomycin Interaction with a Strongly Bound Hairpin DNA Substrate, and Implications for Cleavage of the Bound DNA

Cited by 19 publications

References 45 publications

Hairpin DNA Sequences Bound Strongly by Bleomycin Exhibit Enhanced Double-Strand Cleavage

Hairpin DNA Sequences Bound Strongly by Bleomycin Exhibit Enhanced Double-Strand Cleavage

Characterization of Bleomycin-Mediated Cleavage of a Hairpin DNA Library

A Short DNA Sequence Confers Strong Bleomycin Binding to Hairpin DNAs

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