14-3-3ε Mediates the Cell Fate Decision-Making Pathways in Response of Hepatocellular Carcinoma to Bleomycin-Induced DNA Damage

Background

Lack of understanding of the response of hepatocellular carcinoma (HCC) to anticancer drugs causes the high mortality of HCC patients. Bleomycin (BLM) that induces DNA damage is clinically used for cancer therapy, while the mechanism underlying BLM-induced DNA damage response (DDR) in HCC cells remains ambiguous. Given that 14-3-3 proteins are broadly involved in regulation of diverse biological processes (BPs)/pathways, we investigate how a 14-3-3 isoform coordinates particular BPs/pathways in BLM-induced DDR in HCC.

Methodology/Principal Findings

Using dual-tagging quantitative proteomic approach, we dissected the 14-3-3ε interactome formed during BLM-induced DDR, which revealed that 14-3-3ε via its associations with multiple pathway-specific proteins coordinates multiple pathways including chromosome remodeling, DNA/RNA binding/processing, DNA repair, protein ubiquitination/degradation, cell cycle arrest, signal transduction and apoptosis. Further, “zoom-in” investigation of the 14-3-3ε interacting network indicated that the BLM-induced interaction between 14-3-3ε and a MAP kinase TAK1 plays a critical role in determining cell propensity of apoptosis. Functional characterization of this interaction further revealed that BLM triggers site-specific phosphorylations in the kinase domain of TAK1. These BLM-induced changes of phosphorylations directly correlate to the strength of the TAK1 binding to 14-3-3ε, which govern the phosphorylation-dependent TAK1 activation. The enhanced 14-3-3ε-TAK1 association then inhibits the anti-apoptotic activity of TAK1, which ultimately promotes BLM-induced apoptosis in HCC cells. In a data-dependent manner, we then derived a mechanistic model where 14-3-3ε plays the pivotal role in integrating diverse biological pathways for cellular DDR to BLM in HCC.

Conclusions

Our data demonstrated on a systems view that 14-3-3ε coordinates multiple biological pathways involved in BLM-induced DDR in HCC cells. Specifically, 14-3-3ε associates with TAK1 in a phosphorylation-dependent manner to determine the cell fate of BLM-treated HCC cells. Not only individual proteins but also those critical links in the network could be the potential targets for BLM-mediated therapeutic intervention of HCC.     

Effects of hypoxanthine substitution on bleomycin-mediated DNA strand degradation in DNA-RNA hybrids.

We have reported on the differences in site-specific cleavage between DNA and DNA-RNA hybrids by various prototypic DNA cleavers (accompanying paper). In the case of bleomycin (BLM), degradation at 5'-GC-3'sites was suppressed relative to the same sequence in double-stranded DNA, while 5'-GT-3' damage remained constant. We now present results of our further investigation on the chemical and conformational factors that contribute to BLM-mediated DNA strand cleavage of DNA-RNA hybrids. Substitution of guanine by hypoxanthine on the RNA strand of hybrids resulted in a significant enhancement of 5'-GC-3' site damage on the DNA strand relative to double-stranded DNA, thus reversing the suppression noted at these sites. Additionally, 5'-AT-3' sites, which are damaged significantly more in the hybrid than in DNA, exhibit decreased product formation when hypoxanthine is present on the RNA strand of hybrids. However, when hypoxanthine is substituted for guanine on the DNA strand (a GC cleavage site becomes IC), 5'-IT-3' and 5'-IC-3' site cleavage is almost completely suppressed, whereas AT site cleavage is dramatically enhanced. The priority in metallobleomycin site-specific cleavage of hybrids changes with hypoxanthine substitution: the cleavage priority is AT > GT > GC in native hybrid; GC > GT > AT in hybrids substituted with hypoxanthine in the RNA strand; AT >> GT approximately GC in hybrids substituted with hypoxanthine in the DNA strand. The results of kinetic isotope effect studies on BLM cleavage are presented and, in most cases, the values are larger for the hypoxanthine-substituted hybrid. The results suggest that the 2-amino groups of guanine residues on both strands of the nucleic acid play an important role in modulation of the binding and cleavage specificity of BLM.     

The genome-wide sequence specificity of DNA cleavage by bleomycin analogues in human cells

Bleomycin (BLM) is a cancer chemotherapeutic agent that cleaves cellular DNA at specific sequences. Using next-generation Illumina sequencing, the genome-wide sequence specificity of DNA cleavage by two BLM analogues, 6′-deoxy-BLM Z and zorbamycin (ZBM), was determined in human HeLa cells and compared with BLM. Over 200 million double-strand breaks were examined for each sample, and the 50,000 highest intensity cleavage sites were analysed. It was found that the DNA sequence specificity of the BLM analogues in human cells was different to BLM, especially at the cleavage site (position “0”) and the “+1” position. In human cells, the 6′-deoxy-BLM Z had a preference for 5′-GTGY*MC (where * is the cleavage site, Y is C or T, M is A or C); it was 5′-GTGY*MCA for ZBM; and 5′-GTGT*AC for BLM. With cellular DNA, the highest ranked tetranucleotides were 5′-TGC*C and 5′-TGT*A for 6′-deoxy-BLM Z; 5′-TGC*C, 5′-TGT*A and 5′-TGC*A for ZBM; and 5′-TGT*A for BLM. In purified human genomic DNA, the DNA sequence preference was 5′-TGT*A for 6′-deoxy-BLM, 5′-RTGY*AYR (where R is G or A) for ZBM, and 5′-TGT*A for BLM. Thus, the sequence specificity of the BLM analogue, 6′-deoxy-BLM Z, was similar to BLM in purified human DNA, while ZBM was different.     

Effect of dimethyl sulfides on the induction of apoptosis in human leukemia Jurkat cells and HL-60 cells

Organosulfur compounds have been established to possess anticancer effects. To provide a better understanding of the biological function of dimethyl sulfides, dimethyl monosulfide (Me(2)S), dimethyl disulfide (Me(2)S(2)), dimethyl trisulfide (Me(2)S(3)) and dimethyl tetrasulfide (Me(2)S(4)) were used as experimental materials to investigate their effects on apoptosis induction in human leukemia Jurkat cells and HL-60 cells. Treatment with 20 muM dimethyl sulfides for 24 h decreased the viability of both cells. The cell viability-reducing effect of these sulfides was in the following order: Me(2)S(4) asymptotically equal to Me(2)S(3) > Me(2)S(2) asymptotically equal to Me(2)S for Jurkat cells and Me(2)S(4) > Me(2)S(3) > Me(2)S(2) asymptotically equal to Me(2)S for HL-60 cells. Me(2)S(3) and Me(2)S(4) significantly induced DNA fragmentation and caspase-3 activation. The addition of GSH or NAC completely suppressed the sulfide-induced apoptosis. Our results indicate that dimethyl sulfides with a larger number of sulfur atoms more strongly induced apoptosis in both human leukemia cells via ROS production and caspase-3 activation.     

Novel peptide derivatives of bleomycin A5: synthesis, antitumor activity and interaction with DNA

A series of novel amino acid and peptide derivatives of bleomycin (BLM) A(5) were synthesized. All the compounds possessed significant antitumor activities in vitro against HL-60, BGC-823, PC-3MIE8, and MDA-MB-435 cell lines. Their antitumor activities against MDA-MB-435 were 10-fold higher than BLM A5. The DNA cleavage studies indicated that the hydrophobic amino acid or peptide derivatives of BLM A5 could induce higher cleavage ratio of double to single strand DNA than BLM A5. From the DNA binding studies, we found that the derivatives containing either D-conformation amino acid or basic amino acid could facilitate DNA binding of BLM.     

BLAP75, an essential component of Bloom's syndrome protein complexes that maintain genome integrity

Bloom's syndrome (BS) is a rare human genetic disorder characterized by dwarfism, immunodeficiency, genomic instability and cancer predisposition. We have previously purified three complexes containing BLM, the helicase mutated in this disease. Here we demonstrate that BLAP75, a novel protein containing a putative OB-fold nucleic acid binding domain, is an integral component of BLM complexes, and is essential for their stability in vivo. Consistent with a role in BLM-mediated processes, BLAP75 colocalizes with BLM in subnuclear foci in response to DNA damage, and its depletion impairs the recruitment of BLM to these foci. Depletion of BLAP75 by siRNA also results in deficient phosphorylation of BLM during mitosis, as well as defective cell proliferation. Moreover, cells depleted of BLAP75 display an increased level of sister-chromatid exchange, similar to cells depleted of BLM by siRNA. Thus, BLAP75 is an essential component of the BLM-associated cellular machinery that maintains genome integrity.     

Cleavage of the Bloom's syndrome gene product during apoptosis by caspase-3 results in an impaired interaction with topoisomerase IIIalpha

In higher eukaryotes, the integration of signals triggered in response to certain types of stress can result in programmed cell death. Central to these events is the sequential activation of a cascade of proteinases known as caspases. The final activated effector caspases of this cascade digest a number of cellular proteins, in some cases increasing their enzymatic activity, in others destroying their function. Of the proteins shown to be targets for caspase-mediated proteolysis, a surprisingly large proportion are proteins involved in the signalling or repair of DNA damage. Here we investigate whether BLM, the product of the gene mutated in Bloom’s syndrome, a human autosomal disease characterised by cancer predisposition and sunlight sensitivity, is cleaved during apoptosis. BLM interacts with topoisomerase IIIα and has been proposed to play an important role in maintaining genomic integrity through its roles in DNA repair and replication. We show that BLM is cleaved during apoptosis by caspase-3 and reveal that the main cleavage site is located at the junction between the N-terminal and central helicase domains of BLM. Proteolytic cleavage by caspase-3 produces a 120 kDa fragment, which contains the intact helicase domain and three smaller fragments, the relative amounts of which depend on time of incubation with caspase-3. The 120 kDa fragment retains the helicase activity of the intact BLM protein. However, its interaction with topoisomerase IIIα is severely impaired. Since the BLM–topoisomerase interaction is believed to be necessary for many of the replication and recombination functions of BLM, we suggest that caspase-3 cleavage of BLM could alter the localisation and/or function of BLM and that these changes may be important in the process of apoptosis.     

ATP-dependent G-quadruplex unfolding by Bloom helicase exhibits low processivity

Various helicases and single stranded DNA (ssDNA) binding proteins unfold G-quadruplex (GQ) structures. However, the underlying mechanisms of this activity have only recently come to focus. We report kinetic studies on Bloom (BLM) helicase and human telomeric GQ interactions using single-molecule Förster resonance energy transfer (smFRET). Using partial duplex DNA (pdDNA) constructs with different 5′ ssDNA overhangs, we show that BLM localizes in the vicinity of ssDNA/double-stranded DNA (dsDNA) junction and reels in the ssDNA overhang in an ATP-dependent manner. A comparison of DNA constructs with or without GQ in the overhang shows that GQ unfolding is achieved in 50–70% of reeling attempts under physiological salt and pH conditions. The unsuccessful attempts often result in dissociation of BLM from DNA which slows down the overall BLM activity. BLM-mediated GQ unfolding is typically followed by refolding of the GQ, a pattern that is repeated several times before BLM dissociates from DNA. BLM is significantly less processive compared to the highly efficient GQ destabilizer Pif1 that can repeat GQ unfolding activity hundreds of times before dissociating from DNA. Despite the variations in processivity, our studies point to possible common patterns used by different helicases in minimizing the duration of stable GQ formation.     

RecQ-core of BLM unfolds telomeric G-quadruplex in the absence of ATP

Various helicases and single-stranded DNA (ssDNA) binding proteins are known to destabilize G-quadruplex (GQ) structures, which otherwise result in genomic instability. Bulk biochemical studies have shown that Bloom helicase (BLM) unfolds both intermolecular and intramolecular GQ in the presence of ATP. Using single molecule FRET, we show that binding of RecQ-core of BLM (will be referred to as BLM) to ssDNA in the vicinity of an intramolecular GQ leads to destabilization and unfolding of the GQ in the absence of ATP. We show that the efficiency of BLM-mediated GQ unfolding correlates with the binding stability of BLM to ssDNA overhang, as modulated by the nucleotide state, ionic conditions, overhang length and overhang directionality. In particular, we observed enhanced GQ unfolding by BLM in the presence of non-hydrolysable ATP analogs, which has implications for the underlying mechanism. We also show that increasing GQ stability, via shorter loops or higher ionic strength, reduces BLM-mediated GQ unfolding. Finally, we show that while WRN has similar activity as BLM, RecQ and RECQ5 helicases do not unfold GQ in the absence of ATP at physiological ionic strength. In summary, our study points to a novel and potentially very common mechanism of GQ destabilization mediated by proteins binding to the vicinity of these structures.     

Multimeric BLM is dissociated upon ATP hydrolysis and functions as monomers in resolving DNA structures

Bloom (BLM) syndrome is an autosomal recessive disorder characterized by an increased risk for many types of cancers. Previous studies have shown that BLM protein forms a hexameric ring structure, but its oligomeric form in DNA unwinding is still not well clarified. In this work, we have used dynamic light scattering and various stopped-flow assays to study the active form and kinetic mechanism of BLM in DNA unwinding. It was found that BLM multimers were dissociated upon ATP hydrolysis. Steady-state and single-turnover kinetic studies revealed that BLM helicase always unwound duplex DNA in the monomeric form under conditions of varying enzyme and ATP concentrations as well as 3′-ssDNA tail lengths, with no sign of oligomerization being discerned. Measurements of ATPase activity further indicated that BLM helicase might still function as monomers in resolving highly structured DNAs such as Holliday junctions and D-loops. These results shed new light on the underlying mechanism of BLM-mediated DNA unwinding and on the molecular and functional basis for the phenotype of heterozygous carriers of BLM syndrome.     

Synthesis and SAR studies of 5-(pyridin-4-yl)-1,3,4-thiadiazol-2-amine derivatives as potent inhibitors of Bloom helicase

Human cells utilize a variety of complex DNA repair mechanisms in order to combat constant mutagenic and cytotoxic threats from both exogenous and endogenous sources. The RecQ family of DNA helicases, which includes Bloom helicase (BLM), plays an important function in DNA repair by unwinding complementary strands of duplex DNA as well as atypical DNA structures such as Holliday junctions. Mutations of the BLM gene can result in Bloom syndrome, an autosomal recessive disorder associated with cancer predisposition. BLM-deficient cells exhibit increased sensitivity to DNA damaging agents indicating that a selective BLM inhibitor could be useful in potentiating the anticancer activity of these agents. In this work, we describe the medicinal chemistry optimization of the hit molecule following a quantitative high-throughput screen of >355,000 compounds. These efforts lead to the identification of ML216 and related analogs, which possess potent BLM inhibition and exhibit selectivity over related helicases. Moreover, these compounds demonstrated cellular activity by inducing sister chromatid exchanges, a hallmark of Bloom syndrome.     

The Bloom's syndrome helicase can promote the regression of a model replication fork

Homozygous inactivation of BLM gives rise to Bloom's syndrome, a disorder associated with genomic instability and cancer predisposition. BLM encodes a member of the RecQ DNA helicase family that is required for the maintenance of genome stability and the suppression of sister-chromatid exchanges. BLM has been proposed to function in the rescue of replication forks that have collapsed or stalled as a result of encountering lesions that block fork progression. One proposed mechanism of fork rescue involves regression in which the nascent leading and lagging strands anneal to create a so-called "chicken foot" structure. Here we have developed an in vitro system for analysis of fork regression and show that BLM, but not Escherichia coli RecQ, can promote the regression of a model replication fork. BLM-mediated fork regression is ATP-dependent and occurs processively, generating regressed arms of >250 bp in length. These data establish the existence of a eukaryotic protein that could promote replication fork regression in vivo and suggest a novel pathway through which BLM might suppress genetic exchanges.     

Selective cleavage of BLM, the bloom syndrome protein, during apoptotic cell death

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a high incidence of cancer and genomic instability. BLM, the protein defective in BS, is a RECQ-like helicase that is presumed to function in mammalian DNA replication, recombination, or repair. We show here that BLM, but not the related RECQ-like helicase WRN, is rapidly cleaved in cells undergoing apoptosis. BLM was cleaved to 47- and 110-kDa major fragments, with kinetics similar to the apoptotic cleavage of poly(A)DP-ribose polymerase. BLM cleavage was prevented by a caspase 3 inhibitor and did not occur in caspase 3-deficient cells. Moreover, recombinant BLM was cleaved to 47- and 110-kDa fragments by caspase 3, but not caspase 6, in vitro. The caspase 3 recognition sequence (412)TEVD(415) was verified by mutating aspartate 415 to glycine and showing that this mutation rendered BLM resistant to caspase 3 cleavage. Cleavage did not abolish the BLM helicase activity but abolished BLM nuclear foci and the association of BLM with condensed DNA and the insoluble matrix. The results suggest that BLM, but not WRN, is an early selected target during the execution of apoptosis.     

Amplification of C1027-induced DNA cleavage and apoptosis by a quinacrine-netropsin hybrid molecule in tumor cell lines

We examined the effect of a newly synthesized DNA-binding ligand, quinacrine-netropsin hybrid molecule (QN), on cytotoxicity, apoptosis, and DNA strand breaks induced by an enediyne antitumor antibiotic, C1027. QN significantly enhanced C1027-induced cellular DNA strand breaks, caspase-3 activation, and DNA ladder formation, characteristic of apoptosis, in human HL-60 cells. Flow cytometry revealed that C1027-induced intracellular H(2)O(2) generation was enhanced by QN, suggesting that QN enhances C1027-induced cytotoxic effect through H(2)O(2)-mediated apoptosis. QN also significantly enhanced C1027-induced apoptosis in BJAB cells, and the inhibition of apoptosis was observed in BJAB cells transfected with Bcl-2 gene. The experiment using (32)P-labeled DNA fragments showed that the addition of QN enhanced C1027-induced double-stranded DNA cleavage at the 5'-AGG-3'/3'-TCC-5' sequence (cutting sites are underlined). These results suggest that QN enhances C1027-induced antitumor effect via DNA cleavage and apoptosis. The present study shows a novel approach to the potentially effective anticancer therapy.     

ATR and ATM-Dependent Movement of BLM Helicase during Replication Stress Ensures Optimal ATM Activation and 53BP1 Focus Formation

The BLM helicase, a deficiency in which markedly increases cancer incidence in humans, isrequired for optimal repair during DNA replication. We show that BLM rapidly moves fromPML nuclear bodies to damaged replication forks, returning to PML bodies several hours later,owing to activities of the DNA damage response kinases ATR and ATM, respectively.Immunofluorescence and cellular fractionation demonstrate that BLM partitions to different subcellularcompartments after replication stress. Unexpectedly, fibroblasts lacking BLM weredeficient in phospho-ATM (S-1981) and 53-binding protein-1 (53BP1), and these proteins failedto form foci following replication stress. Expression of a dominant p53 mutant or helicasedeficientBLM restored replication stress-induced 53BP1 foci, but only mutant p53 restoredoptimal ATM activation. Thus, optimal repair of damaged replication fork lesions likelyrequires both ATR and ATM, BLM recruits 53BP1 to these lesions independent of its helicaseactivity, and optimal activation of ATM requires both p53 and BLM helicase activities.

Supplemental material for this paper can be found at the following link:

http://www.landesbioscience.com/journals/cc/davalosCC3-12-sup.pdf     

Copper(II) facilitates bleomycin-mediated unwinding of plasmid DNA

The unwinding of plasmid DNA by bleomycin A2 (BLM A2) was investigated by use of two-dimensional gel electrophoresis. It was found that Cu2+ ions greatly facilitated the unwinding of topoisomers of plasmid DNA by BLM A2 at concentrations where cupric ions alone had no effect on DNA supercoiling. The concentration of BLM A2 required for observable unwinding was reduced at least 100-fold in the presence of equimolar Cu2+. A plot of [Cu2+] vs extent of DNA unwinding in the presence of 10(-4) M BLM A2 gave a curve consistent with the action of cupric ions on BLM in an allosteric fashion, possibly rearranging the drug into a conformation that facilitates DNA unwinding. The participation of the metal center in enhancing DNA unwinding via direct ionic interaction with one or more negatively charged groups on the DNA duplex also seems possible. Further analysis of the structural factors required for BLM-mediated DNA unwinding was carried out with Cu2+ + BLM demethyl A2, the latter of which differs from BLM A2 only in that it lacks a methyl group, and associated positive charge, at the C-terminus. Cu(II).BLM demethyl A2 was found to be much less effective than Cu(II).BLM A2 as a DNA unwinding agent, emphasizing the strong dependence of this process on the presence of positively charged groups within the BLM molecule. These findings constitute the first direct evidence that the metal center of BLM can participate in DNA interaction, as well as in the previously recognized role of oxygen binding and activation.     

The geometry of DNA supercoils modulates the DNA cleavage activity of human topoisomerase I

Human topoisomerase I plays an important role in removing positive DNA supercoils that accumulate ahead of replication forks. It also is the target for camptothecin-based anticancer drugs that act by increasing levels of topoisomerase I-mediated DNA scission. Evidence suggests that cleavage events most likely to generate permanent genomic damage are those that occur ahead of DNA tracking systems. Therefore, it is important to characterize the ability of topoisomerase I to cleave positively supercoiled DNA. Results confirm that the human enzyme maintains higher levels of cleavage with positively as opposed to negatively supercoiled substrates in the absence or presence of anticancer drugs. Enhanced drug efficacy on positively supercoiled DNA is due primarily to an increase in baseline levels of cleavage. Sites of topoisomerase I-mediated DNA cleavage do not appear to be affected by supercoil geometry. However, rates of ligation are slower with positively supercoiled substrates. Finally, intercalators enhance topoisomerase I-mediated cleavage of negatively supercoiled substrates but not positively supercoiled or linear DNA. We suggest that these compounds act by altering the perceived topological state of the double helix, making underwound DNA appear to be overwound to the enzyme, and propose that these compounds be referred to as ‘topological poisons of topoisomerase I’.     

敲减BLM解旋酶表达增强前列腺癌PC3细胞对丝裂霉素C的敏感性

丝裂霉素C是一种广谱抗肿瘤抗生素,对多种癌症有抗癌作用,其作用原理可使细胞的DNA发生链间交联,引起DNA双链断裂,阻碍DNA的复制,从而抑制肿瘤细胞分裂。临床上主要用于胃癌、肠癌、肝癌及胰腺癌等消化道癌方面的治疗。本文研究丝裂霉素C对转染人BLM解旋酶基因（shRNA载体）前后前列腺癌PC3细胞活性的影响。使用前期成功构建的干扰载体转染PC3细胞,在转染48 h后加药,通过荧光定量PCR、MTT法、Transwell小室实验、细胞划痕实验、流式细胞术,分别检测加药12、24、36 h BLM基因的表达量、PC3细胞增殖能力、侵袭能力、迁移能力及凋亡情况的变化。结果显示,敲减BLM基因表达后的PC3细胞相对于正常PC3细胞其增殖能力、侵袭能力和迁移能力能显著被丝裂霉素C抑制,且丝裂霉素C能显著促进其细胞的凋亡,说明BLM基因低表达的前列腺癌细胞对丝裂霉素C更敏感。研究结果为丝裂霉素C在前列腺癌的临床治疗上奠定了理论基础。     

Absence of p53 enhances growth defects and etoposide sensitivity of human cells lacking the Bloom syndrome helicase BLM

The Bloom syndrome helicase BLM and the tumor-suppressor protein p53 play important roles in preserving genome integrity. Here, we knock out the genes for BLM and p53 in a human pre-B-cell line, Nalm-6. We show that p53 plays an important role in cell proliferation, but not apoptosis, when BLM is absent. Intriguingly, despite the apoptotic function of p53, BLM(/)TP53(/) cells were more sensitive than either single mutant to etoposide, an anticancer agent that poisons DNA topoisomerase II. Our results suggest a direct, BLM-independent role for p53 in etoposide-induced, topoisomerase II-mediated DNA damage in human cells.     

Mechanistic studies on bleomycin-mediated DNA damage: multiple binding modes can result in double-stranded DNA cleavage

The bleomycins (BLMs) are a family of natural glycopeptides used clinically as antitumor agents. In the presence of required cofactors (Fe²⁺ and O₂), BLM causes both single-stranded (ss) and double-stranded (ds) DNA damage with the latter thought to be the major source of cytotoxicity. Previous biochemical and structural studies have demonstrated that BLM can mediate ss cleavage through multiple binding modes. However, our studies have suggested that ds cleavage occurs by partial intercalation of BLM's bithiazole tail 3′ to the first cleavage site that facilitates its re-activation and re-organization to the second strand without dissociation from the DNA where the second cleavage event occurs. To test this model, a BLM A5 analog (CD-BLM) with β-cyclodextrin attached to its terminal amine was synthesized. This attachment presumably precludes binding via intercalation. Cleavage studies measuring ss:ds ratios by two independent methods were carried out. Studies using [³²P]-hairpin technology harboring a single ds cleavage site reveal a ss:ds ratio of 6.7 ± 1.2:1 for CD-BLM and 3.4:1 and 3.1 ± 0.3:1 for BLM A2 and A5, respectively. In contrast with BLM A5 and A2, however, CD-BLM mediates ds-DNA cleavage through cooperative binding of a second CD-BLM molecule to effect cleavage on the second strand. Studies using the supercoiled plasmid relaxation assay revealed a ss:ds ratio of 2.8:1 for CD-BLM in comparison with 7.3:1 and 5.8:1, for BLM A2 and A5, respectively. This result in conjunction with the hairpin results suggest that multiple binding modes of a single BLM can lead to ds-DNA cleavage and that ds cleavage can occur using one or two BLM molecules. The significance of the current study to understanding BLM's action in vivo is discussed.