the antitumor agent 
 bleomycin ( blm ) is employed clinically for 
 the treatment of squamous cell carcinomas and malignant lymphomas .
the clinical mixture of bleomycins known as blenoxane 
 consists mainly of blm a2 and blm b2 , and blm a5 is also used as a single 
 chemotherapeutic agent ( figure 1 ) . while these agents are administered therapeutically 
 in metal - free form , it is believed that feblm , or possibly cublm , formed in situ is actually responsible for the observed single- 
 and double - strand damage to dna .
double - strand 
 dna cleavage by feblm occurs at a frequency greater than what 
 could be anticipated based on the random accumulation of single - strand 
 breaks and has often been suggested to 
 form the basis for the antitumor activity of bleomycin .
chemical structures 
 of bleomycins a2 , b2 , 
 and a5 . while the ability of 
 bleomycin to mediate double - strand dna cleavage 
 is believed to form the basis for its antitumor activity , the specific 
 lesion(s ) that lead to tumor cell killing
recently , we have reported that a library of 10 hairpin dnas selected for their ability to bind tightly to 
 blm exhibited greatly enhanced double - strand cleavage ( figure s1 ) .
the majority 
 of double - strand cleavage events were of a novel type involving two 
 independent single - strand cleavages , rather 
 than the single coupled double - strand cleavage described earlier .
further , it was noted that the number of double - strand cleavages 
 of a given hairpin dna seemed to be in direct proportion to the affinity 
 of that dna for fe(ii)blm a5 . to obtain greater insight into the structural features in 
 the hairpin 
 dnas which enabled their tight binding to bleomycin
, we prepared a 
 new 64-nucleotide ( nt ) hairpin dna library containing eight randomized 
 base pairs .
the strategy employed was the same as that reported previously , with the exception that the selection for tight 
 binders was carried out using immobilized fe(iii)blm a5 rather than metal free blm a5 .
thirty hairpin dnas were 
 isolated from this library and sequenced ; none of these had the same 
 sequence as the 10 dnas in the original library .
careful inspection of the 10 dnas studied from the original 
 hairpin dna library revealed that the two dnas which bound most strongly 
 to fe(ii)blm a5 ( dnas 2 and 7 ) shared the common sequence 5-acgc/5-gcgt , albeit 
 not in the same position within the dnas ( figure 2 ) .
accordingly , the 30 newly identified hairpin dnas were 
 inspected to determine whether any of them also had the sequence 5-acgc/5-gcgt . 

in fact two such dnas ( 12 and 13 ) were identified 
 ( figure 2 ) and formed the basis for the current 
 study .
the new dnas were found to bind to bleomycin more tightly 
 than 
 any species identified to date , as judged both by a competition assay 
 and by the use of surface plasmon resonance .
the latter technique gave results for fe(iii)blm b2 binding which were an excellent fit for a 1:1 binding model , arguing 
 for a single , unique site of binding to each hairpin dna .
in spite 
 of the unique binding site , dna 12 underwent double - strand 
 cleavage at five sites by fe(ii)blm a5 , and dna 13 also gave five sets of double - strand cleavage products . 

while the number of double - strand dna cleavage sites did not increase 
 beyond that noted in our earlier study , 9 of the 10 double - strand 
 cleavage sites in dnas 12 and 13 resulted 
 from two closely spaced but independent single strand cleavage events . to provide further evidence for the involvement of the sequence 
 5-acgc/5-gcgt in blm binding , we designed a hairpin 
 dna ( 14 ) in which the initially randomized eight base - pair 
 sequence was 5-tttttttt/5-aaaaaaaa ( figure 2 ) .
while this hairpin dna had poor affinity for 
 fe(ii)blm a5 as anticipated , replacement of the central 
 four base pairs with 5-acgc/5-gcgt resulted in a hairpin 
 dna ( 15 ) which had dramatically enhanced affinity for 
 fe(ii)blm a5 .
on the basis of the results obtained 
 with hairpin dnas containing 
 the motif 5-acgc/5-gcgt , we suggest a possible mechanism 
 for tumor cell killing by bleomycin .
as shown 
 in figure 3 and table 1 , hairpin dnas 2 and 7 were capable of 
 binding strongly to fe(ii)blm a5 as reported previously , thereby potently 
 suppressing the cleavage of a 16-nt profluorescent hairpin dna present 
 at an equimolar concentration .
each of these 64-nt hairpin dnas inhibited 
 cleavage of the profluorescent hairpin dna to the extent of 97% ( table 1 ) , as reported previously .
newly selected hairpin dnas 12 and 13 bound 
 to fe(ii)blm a5 even more strongly , suppressing 99 
 and 98% , respectively , of cleavage of the profluorescent hairpin dna 
 ( figure 3 and table 1 ) .
the binding specificities of dnas 7 , 12 and 13 for fe(ii)blm b2 were also 
 determined and found to be quite similar ( 97 , 97 , and 99% , respectively ) 
 ( table 1 ) .
fluorescence emission spectra resulting 
 from treatment of 16-nt 
 hairpin dna - cf15 with fe(ii)blm a5 in 
 the presence or absence of 64-nt hairpin dnas .
the reaction mixture 
 contained 0.72 m fe(ii)blm a5 , 0.72 m 
 hairpin dna - cf15 , and 0.72 m hairpin dna 2 , 7 , 12 , or 13 in 10 mm na 
 cacodylate buffer solution , ph 7.0 , containing 100 mm nacl .
the fluorescence 
 emission spectra were obtained following excitation at 310 nm and 
 25 c .
also shown is the fluoresence emission spectrum of the 
 16-nt hairpin dna - cf15 treated with blm a5 in 
 the absence of any 64-nt hairpin dna under the same conditions (  no 
 competitor  ) .
the binding 
 specificity ( % ) was 
 calculated as the decrease in fluorescence intensity at maximum emission 
 wavelength ( 450 nm ) from no competitor ( 0% ) through the reaction mixture 
 without fe ( 100% ) . in order to further quantify the dna binding affinity of feblm , 
 biosensor surface plasmon resonance ( spr ) experiments
were conducted 
 with immobilized hairpin dnas . to compare the sensorgram saturation 
 levels , approximately the same amounts of the hairpin
dnas 7 , 12 , and 13 were immobilized on the surface 
 of each sensor chip .
the equilibrium constants obtained from both 
 global kinetic fitting of the sensorgrams and the steady - state analyses 
 were then fitted to appropriate binding models .
the data are listed 
 in tables 2 and 3 , where 
 they are also compared with the values determined previously for hairpin 
 dna 2 .
10 , 20 , 40 , 70 , 
 and 90 nm concentrations 
 of fe(iii)blm b2 were used to determine the kinetic 
 rate constants .
error values given in the table were obtained during 
 the fitting of data with biacore t200 evaluation software . based on 
 reproducibility of results ,
it is clear from the shape of the binding curves of fe(iii)blm 
 b2 with three different hairpin dnas that the on- and off - rates 
 vary with changes in the position of the acgc / gcgt and flanking sequences 
 ( figures 4 and 5 ) .
initially 
 the ka values for fe(iii)blm b2 were determined for hairpin dnas 7 , 12 , and 13 at 15 c and 10 mm nacl . under these conditions , 
 the strongest binding
was observed for the fe(iii)blm b2 complex with hairpin dna 13 ( table 2 ) . the on - rate constant ka was determined as 2.3  10 m s , while the off - rate kd was quite low ( 0.0075 s ) ( table 2 ) which makes this sequence the strongest fe(iii)blm 
 b2 binder in this set ( ka 30.7 
  10 m ) ( figure 4 ) . under the same conditions fe(iii)blm b2 bound to hairpin dnas 7 and 12 with almost 
 equal affinity ( 17.0  10 and 17.3  10 m , respectively ; table 2 ) .
thus , the relative affinities of fe(iii)blm b2 with three hairpin dnas were found to be dna 13 > dna 12  dna 7 ( table 2 ) .
spr sensorgrams for the interaction of fe(iii)blm 
 b2 with ( a ) hairpin dna 7 , ( b ) hairpin dna 12 , and ( c ) hairpin dna 13 at 10 mm nacl concentrations 
 and 15 c .
the individual sensorgrams ( colored ) represent responses 
 at fe(iii)blm b2 concentrations of 10 , 20 , 40 , 70 , 
 and 90 nm ( bottom to top ) . global kinetic fit ( black solid lines ) 
 with a 1:1 model was performed using biacore t200 evaluation software 
 to obtain kinetic association and dissociation rate constants .
spr sensorgrams for the interaction of fe(iii)blm 
 b2 with ( a ) hairpin dna 7 , ( b ) hairpin dna 12 , and ( c ) hairpin dna 13 at 10 mm nacl concentrations 
 and 15 c .
the individual sensorgrams ( colored ) represent responses 
 at fe(iii)blm b2 concentrations of 100 , 200 , 300 , 
 500 , 700 , and 900 nm ( bottom to top ) . global kinetic fit ( black solid 
 lines ) with a 1:1 model was performed using biacore t200 evaluation 
 software to obtain kinetic association and dissociation rate constants .
spr experiments involving fe(iii)blm 
 b2 with all 
 three hairpin dna sequences were also conducted at 25 c and 
 10 mm salt concentration . under these conditions
the relative affinities 
 of the three hairpin dnas for fe(iii)blm b2 were 
 unchanged ( table 3 ) .
it is readily apparent 
 from the steady - state fit data ( figure 6 ) that 
 the temperature had a significant effect on the absolute binding affinities 
 ( tables 2 and 3 ) ; fe(iii)blm 
 b2 exhibited 34-fold weaker binding for all three 
 hairpin dnas with increasing temperature ( figure 6 ; tables 2 and 3 ) .
spr equilibrium binding plots of fe(iii)blm b2 with 
 ( a ) hairpin dna 7 ,
( b ) hairpin dna 12 , and 
 ( c ) hairpin dna 13 at 10 mm nacl concentrations 
 and 15 ( red ) and 25 ( blue ) c .
the steady - state response values 
 were fitted as a function of free ligand concentration to a single - site 
 interaction model .
the binding affinities are listed in tables 2 and table 3 it may be noted that hairpin dnas 7 , 12 , and 13 all bound to fe(iii)blm 
 b2 with significantly greater affinities than did hairpin 
 dna 2 ( tables 2 and 3 ) in spite of seemingly small differences as measured by the 
 competition 
 assay ( table 1 ) .
the steady - state response 
 values were fitted as a function of free ligand concentration to a 
 single - site interaction model . in the present case
, a 1:1 affinity 
 model provided an excellent fit , quite consistent with the kinetic 
 fitting data . in comparison , for hairpin dna 2 , the steady - state 
 data indicated the presence of at least one and probably multiple 
 weaker binding sites for fe(iii)blm b2 . while the competition assay was less useful 
 than the spr assay in differentiating between dnas 7 , 12 , and 13 , it was essential for quantifying 
 the binding of dna 14 ( vide infra ) , 
 which is not bound strongly enough to be analyzed by spr .
the competition 
 assay also employs fe(ii)blm ( as compared with fe(iii)blm 
 in the spr assay ) , thus measuring the behavior of the species actually 
 involved in dna cleavage .
we have recently reported an analysis 
 of double - strand cleavage 
 of a library of 10 hairpin dnas selected for their ability to bind 
 tightly to bleomycin .
the analysis was 
 carried out by modification of the strategy first described by povirk . following treatment of the alternatively 5- 
 and 3-p end - labeled hairpin dnas with
fe(ii)blm 
 a5 , the co - migrating bands isolated from the nondenaturing 
 gels were analyzed on a sequencing gel .
these co - migrating bands should 
 logically represent double - strand cleavage products , differing only 
 in the site of p end - labeling .
in addition , the bands 
 on the native gels that co - migrated with the uncleaved hairpin dna 
 were also analyzed by sequencing gel analysis to identify the location 
 of species of type iii ( figure 7 ) , i.e. , primary 
 sites of cleavage opposite alkali - labile sites . where these type iii 
 species occurred in the same positions as frank double - strand cleavage 
 ( species of type iv , figure 7 ) ,
identification 
 of the strand on which cleavage had occurred to form the type iii 
 lesion permitted unambiguous identification of the primary site of 
 cleavage .
the absence of a unique primary 
 site of cleavage is a hallmark of a double - strand break formed by 
 two closely spaced single - strand cleavages .
mechanisms 
 of double - strand dna cleavage induced by bleomycin . activated feblm abtracts h from 
 c-4 of deoxyribose at a primary cleavage site , producing either 
 an apyrimidinic / apurinic site ( i ) or a single - strand break having 
 a 3-phosphoroglycolate terminus ( ii ) .
although the apurinic 
 site ( i ) does not undergo further reaction , strand break ii is a potential 
 target for a secondary blm cleavage reaction on the opposing dna strand . 

the secondary attack of ( re)activated bleomycin by abstracting the 
 c-4 h atom from the secondary site sugar affords either a 
 double - strand break with 5-phosphate and 3-phosphoroglycolate 
 termini ( iv ) or a strand break at the primary site accompanied by 
 an ap lesion at the secondary site ( iii ) . the latter upon treatment 
 with mild base can produce a double - strand cleavage product ( v ) .
hairpin dnas 12 and 13 were alternatively 
 5- and 3-p end - labeled and then subjected 
 to cleavage by fe(ii)blm a5 ; the results are shown 
 in figures s2 and s3 , respectively .
a total 
 of 16 cleavages were noted for dna 12 and also 16 cleavages 
 for hairpin dna 13 .
the same radiolabeled hairpin 
 dnas were also employed to determine 
 the sites of double - strand cleavage . following treatment with fe(ii)blm 
 a5 ,
as shown in the native gel in figure 8a , in addition to a band that co - migrated with full 
 length hairpin dna , five sets of bands of comparable mobility were 
 apparent in the alternatively 5- and 3-p end - labeled dnas 12 , consistent with five double - strand 
 cleavage events .
the individual bands were recovered from the native 
 gel , and each was subjected to analysis on a sequencing gel ( figure 8b ) .
the sequencing gel revealed the positions of 
 double - strand dna cleavage for each of the recovered bands .
in fact , 
 five double - strand cleavages were observed , including cleavage at 
 t6/t60 , t7/a59 , t10/a55 , t13/a52 , and c18/c48 , as summarized in figure 10 .
the only primary site of cleavage observed was for t13 ( figure 8b , lane 2 ) , indicating that 
 the double - strand cleavage at t13/a52 was a 
 coupled double - strand cleavage event .
the other four double - strand 
 cleavages resulted from independent single - strand breaks .
all sites 
 of cleavage of hairpin dna 12 are also summarized in figure s2 .
analysis of bleomycin - induced double - strand 
 cleavage of hairpin 
 dna 12 .
( a ) double - strand cleavage of [ 3-p]-end - labeled ( lane 2 ) and [ 5-p]-end - labeled 
 ( lane 3 ) 64-nt hairpin dna 12 by fe(ii)bleomycin 
 a5 .
lane 1 , [ 3-p]-end - labeled dna 
 alone ; lane 2 , 1.5 m fe(ii)blm a5 ; lane 3 , 
 1.5 m fe(ii)blm a5 ; lane 4 , [ 5-p]-end - labeled dna alone .
( b ) sequencing gel analysis of 
 fe(ii)bleomycin - induced sites of double - strand cleavage of [ 5-p]-end - labeled ( lanes 17 ) and [ 3-p]-end - labeled ( lanes 814 ) hairpin dna 12 . each 
 lane ( except lanes 1 and 14 ) corresponds to a numbered cleavage band , 
 shown in ( a ) .
gilbert g+a sequencing lane of 
 [ 5-p]-end - labeled dna 12 ; lane 2 , 
 band 3a ; lane 3 , band 3b ; lane 4 , band 3c ; lane 5 , band 3d ; lane 6 , 
 band 3e ; lane 7 , band 3f ; lane 8 , band 2f ; lane 9 , band 2e ; lane 10 , 
 band 2d ; lane 11 , band 2c ; lane 12 , band 2b ; lane 13 , band 2a ; lane 
 14 , maxam  gilbert g+a sequencing lane of [ 3-p]-end - labeled dna 12 .
the same two - stage analysis was carried out for double - strand 
 cleavage 
 of hairpin dna 13 ( figure 9 ) . 
 as shown in the native gel in figure 9a , five 
 sets of co - migrating bands
were observed when the 5- and 3-p end - labeled dnas 13 were run in adjacent lanes 
 on the native gel .
recovery of the bands from the native gel , followed 
 by further analysis of each on a sequencing gel , permitted the positions 
 of double - strand cleavage to be identified .
as anticipated , five double - strand 
 cleavage events were identified , and these are summarized in figure 10 .
the double - strand 
 cleavage sites included t6/t60 , t7/a59 , t10/t56 , c12/c54 and c16/c50 .
no primary site of cleavage 
 was observed , indicating that all five double - strand cleavages were 
 produced as closely spaced single - strand cleavage events .
analysis of bleomycin - induced double - strand cleavage of 
 hairpin 
 dna 13 .
( a ) double - strand cleavage of [ 3-p]-end - labeled ( lane 2 ) and [ 5-p]-end - labeled 
 ( lane 3 ) 64-nt hairpin dna 13 by fe(ii)bleomycin 
 a5 .
lane 1 , [ 3-p]-end - labeled dna 
 alone ; lane 2 , 1.5 m fe(ii)blm a5 ; lane 3 , 
 1.5 m fe(ii)blm a5 ; lane 4 , [ 5-p]-end - labeled dna alone .
( b ) sequencing gel analysis of 
 sites of febleomycin - induced double - strand cleavage of [ 5-p]-end - labeled ( lanes 17 ) and [ 3-p]-end - labeled ( lanes 814 ) hairpin dna 13 . each 
 lane ( except lanes 1 and 14 ) corresponds to a numbered cleavage band , 
 shown in ( a ) .
gilbert g+a sequencing lane of 
 [ 5-p]-end - labeled dna 13 ; lane 2 , 
 band 3a ; lane 3 , band 3b ; lane 4 , band 3c ; lane 5 , band 3d ; lane 6 , 
 band 3e ; lane 7 , band 3f ; lane 8 , band 2f ; lane 9 , band 2e ; lane 10 , 
 band 2d ; lane 11 , band 2c ; lane 12 , band 2b ; lane 13 , band 2a ; lane 
 14 , maxam 
black arrows correspond to coupled 
 double - strand cleavage events , whereas red arrows indicate noncoupled 
 double - strand cleavage events , resulting from two independent single - strand 
 cleavages on opposite strands .
nucleotide colored in red indicates 
 a primary site of double - strand dna cleavage for a coupled double - strand 
 cleavage event . in summary ,
for dna 12 , there was a single site at 
 which double - strand cleavage involved a coupled process , namely t13/a52 .
the remaining four double - strand cleavages each resulted from two 
 independent cleavage events in close proximity . in the case of hairpin 
 dna 13 ,
all five observed double - strand cleavage events 
 resulted from closely spaced single - strand cleavage .
of special 
 interest were the cleavage events involving the 5-acgc/5-gcgt 
 sequences common to hairpin dnas 2 , 7 , 12 , and 13 .
for hairpin dna 2 , these 
 included a15/t50 , c16/c48 , and c18/(t46 ) , all three of which involved 
 coupled double - strand cleavage events .
for hairpin dna 7 , the sites of cleavage included ( t11)/t53 , 
 c13/c51 , and c15/(c49 ) , 
 only two of which involved coupled double - strand cleavage .
for hairpin 
 dna 12 , c18/c48 was the only double - strand 
 cleavage site , and for dna 13 , c16/c50 was the only double - strand cleavage site ; neither of these represented 
 coupled events .
thus , the only common feature of the cleavages within 
 the common 5-acgc/5-gcgt sequences was that the cytidine 
 moiety in the 5-gcgt sequence was cleaved in each case .
the assumption that the 5-acgc/5-gcgt sequence 
 common to hairpin dnas 2 , 7 , 12 , and 13 was responsible for the exceptionally strong 
 binding of these dnas to feblm was tested directly .
the randomized 
 region of the 64-nt hairpin dna was substituted with 5-tttttttt/5-aaaaaaaa 
 ( hairpin dna 14 , figure 2 ) . when 
 this hairpin dna was employed in the competition assay with an equimolar 
 amount of the profluorescent 16-nt hairpin dna ,
the reduction in release 
 of the fluorescent nucleobase from the 16-nt dna was only 21% ( figure 11 and table 1 ) , indicating 
 quite weak binding by fe(ii)blm a5 .
substitution 
 of nucleosides 1316 by 5-acgc ( and concomitant substitution 
 of nucleotides 4952 by 5-gcgt ) afforded hairpin dna 15 .
although the strategy that lead to the preparation of 
 this hairpin dna did not involve any selection whatsoever by an immobilized 
 blm , it was found to have a binding specificity of 93% for fe(ii)blm 
 a5 ( table 1 ) .
fluorescence emission 
 spectra resulting from treatment of 16-nt 
 hairpin dna - cf15 with fe(ii)blm a5 in 
 the presence or absence of 64-nt hairpin dnas 14 and 15 .
the procedure employed was the same as that described 
 in the legend to figure 3 .
the uniqueness of the 5-acgc/5-gcgt motif 
 was studied 
 by searching for other tetranucleotide sequences that occurred multiple 
 times in the two hairpin dna libraries .
a number of sequences occurred 
 at least three times , and seven of these were investigated to identify 
 other motifs associated with strong blm binding .
three of these hairpin dnas 
 were tested for fe(ii)blm a5 binding , and one had 
 a binding specificity of 94% ; however , the binding specificities of 
 the other two were 64% and 86% .
three of these were tested using 
 the competition assay , but the binding specificities of these dnas 
 for fe(ii)blm were relatively modest ( 82% , 64% , and 89% ) .
testing of three of these dnas indicated fe(ii)blm 
 binding specificities of 82% , 64% , and 89% .
other motifs which appeared 
 at least three times included 5-ttga/5-tcaa , 5-ggcc/5-ggcc , 
 5-atcc/5-ggat , and 5-gcgc/5-gcgc . 

the assay of representative hairpin dnas containing these motifs again 
 failed to provide evidence of their ability to confer strong fe(ii)blm 
 binding specificity .
thus , the results obtained with the motif 5-acgc/5-gcgt 
 appeared to be quite unique within these hairpin dna libraries .
the bleomycins are clinically used as antitumor agents whose mechanism 
 of action is believed to involve dna cleavage .
the bleomycins mediate 
 sequence selective dna cleavage , and about 20% of the lesions that 
 they produce involve double - strand dna cleavage .
the double - strand dna breaks have attracted 
 significant attention , as they are presumably difficult to repair 
 at a cellular level and may well form the basis for the antitumor 
 activity of this class of compounds .
the study of double - strand 
 dna cleavage was studied initially by 
 the povirk laboratory , who demonstrated that the most commonly monitored 
 process involved a coupled event in which initial cleavage on one 
 dna strand was followed by oxidative damage in close proximity on 
 the opposite strand . in one study
samples of each of the duplexes were alternatively 5- 
 and 3-p end - labeled , and then treated with fe(ii)blm 
 a2 and separated on a nondenaturing polyacrylamide gel 
 to permit identification of the ( co - migrating ) bands resulting from 
 double - strand cleavage .
this analysis indicated that 
 all of the double - strand breaks involved a 5-g - py sequence 
 on one strand , but there were a variety of sequences involved in the 
 breakage on the opposite strand .
it was assumed that the shared g - py 
 sequence represented the primary cleavage site .
the orientation of 
 the breaks afforded products with blunt ends and 1-nt 5-extensions , 
 but no product with 3-extensions .
in addition to the double - strand breaks , alkali - labile lesions were 
 also noted at the secondary cleavage sites on the strand opposite 
 the primary cleavage site .
the alkali - labile lesion is believed to 
 form by an alternative pathway from the same ( c4 deoxyribose ) 
 radical intermediate that leads to frank strand scission .
critically , because these experiments were carried out under conditions 
 of single - hit kinetics , the authors concluded that the double - strand 
 breaks must have been mediated by a single feblm molecule .
the conclusion that the double - strand breaks were coupled mechanistically 
 has been supported by studies from other laboratories .
this has included 
 the finding of enhanced cleavage opposite an initial blm - induced nick and more detailed studies by absalon et al .
utilizing hairpin dnas bearing an internal p - labeled 
 phosphate group , which enabled measurement of the ratio of single- 
 to double - strand cleavage to be measured at individual sites . an earlier 
 study from our laboratory documented the existence of a highly efficient 
 double - strand cleavage site in a hairpin dna , which was shown to be 
 a coupled event by studying the relative intensities of cleavage at 
 the two sites following alternative 5- or 3-p end - labeling .
all of these studies 
 supported the idea that the double - strand cleavage observed was mediated 
 by a single blm molecule .
analysis of the 10 hairpin dnas revealed 
 the presence of a total 
 of 31 double - strand cleavage sites .
unexpectedly , only 14 of these 
 sites resulted from coupled double - strand cleavage .
the remaining 
 17 double - strand cleavages lacked a unique primary cleavage site and 
 appeared to have arisen from two independent single - strand cleavage 
 events .
the occurrence of this novel type 
 of cleavage was attributed to the exceptional affinity of blm for 
 the hairpin dnas in the library .
consistent with this interpretation 
 was the finding that those dnas in the library which bound most avidly 
 to fe(ii)blm a5 underwent the greatest number of 
 double - strand cleavages .
a study of the dynamic behavior of 
 feblms in the presence 
 of three of these hairpin dnas by surface plasmon resonance also indicated 
 that there was a single strong binding site for each , along with one 
 ( or more likely more than one ) much weaker binding sites . in spite of the single strong binding site , 
 each of these dnas was cleaved strongly at multiple sites , and the 
 fe(ii)blm bound to the hairpin dnas was unavailable for cleavage 
 of a second competitor dna known to be a good substrate for cleavage 
 by feblm in the absence of the 64-nt hairpin dna .
this finding is fully consistent with the observation 
 of double - strand cleavages resulting from independent , closely spaced 
 single - strand cleavage events .
the foregoing studies verified 
 the existence of a number of hairpin 
 dnas able to bind tightly to blm .
these dnas exhibit unusual sequence 
 selectivity of dna cleavage and undergo numerous single- and double - strand 
 cleavage events , the 
 latter of which seem more numerous than in arbitrarily chosen duplex 
 dnas and form via two distinct mechanisms .
however , none of these studies suggested which 
 specific structural elements in the hairpin dnas might be responsible 
 for the strong binding of blm .
inspection of the 10 hairpin dnas employed 
 revealed that the two which bound the most strongly to feblm 
 ( dnas 2 and 7 , figure 1 ) had the common sequence 5-acgc ( and its complement 5-gcgt ) . 

the present study was designed to determine whether this sequence 
 was responsible for the strong binding of hairpin dnas 2 and 7 to blm .
a new randomized library of hairpin 
 dnas was prepared as described 
 previously and used to select additional 
 dnas that bound strongly to blm . in this experiment ,
the selection 
 was carried out using immobilized fe(iii)blm a5 rather 
 than metal free blm a5 .
thirty new hairpin dnas were identified 
 in this selection and sequenced , and two of them ( hairpin dnas 12 and 13 ) were found to contain the sequence 
 5-acgc/5-gcgt .
as shown in figure 3 and table 1 , these two dnas were found 
 to bind more strongly to fe(ii)blm a5 than any previously 
 characterized dna , as judged by a competition binding assay .
the new 
 dnas were also characterized for their interaction with fe(iii)blm 
 b2 by surface plasmon resonance ( figures 46 , tables 2 and 3 ) .
hairpin dna 12 had binding properties not dissimilar to hairpin dna 7 , while hairpin dna 13 clearly bound to fe(iii)blm 
 b2 more avidly than any species tested to date .
interestingly , 
 unlike the hairpin dnas characterized previously , hairpin dnas 12 and 13 gave steady - state 
 response values fully consistent with a 1:1 affinity model .
hairpin dnas 12 and 13 were utilized 
 as substrates for cleavage by fe(ii)blm a5 , and both 
 gave multiple double - strand dna cleavages .
interestingly , unlike 
 the cleavage patterns observed in earlier studies , the 
 orientation of the breaks afforded products with blunt ends and 1-nt 
 3-extensions , the latter of which had not been observed previously 
 as a common feature . in particular ,
hairpin 
 dna 12 gave two double - strand cleavage products with 
 blunt ends and three having 1-nt 3-extensions , while hairpin 
 dna 13 afforded products all of which had 1-nt 3-extensions . 

in fact , analysis of the mechanism of double - strand cleavage indicated 
 that only one of the 10 double - strand cleavage reactions ( involving 
 products with blunt ends ) occurred by a coupled double - strand cleavage 
 mechanism . to determine whether the presence of the 5-acgc/5-gcgt 
 sequence in an arbitrarily chosen hairpin dna sequence would be sufficient 
 to confer strong binding to feblm , we prepared hairpin dna 14 , in which the eight base pair randomized region consisted 
 of a sequence ( 5-tttttttt/5-aaaaaaaa ) which we felt 
 would be unlikely to contribute to efficient binding of feblm . 

in fact , hairpin dna 14 exhibited a binding specificity 
 of only 21% in our competition binding assay ( figure 11 and table 1 ) .
in contrast , when the 
 sequence 5-acgc/5-gcgt was substituted in the middle 
 of this eight base pair region , the binding specificity for fe(ii)blm 
 a5 increased to 93% , providing additional evidence of the 
 importance of this sequence in blm binding . in spite of the 
 importance of the 5-acgc/5-gcgt 
 sequence for blm binding to the hairpin dnas and the spr evidence 
 for unique strong feblm binding sites in dnas 12 and 13
, there was a surprising lack of consistency 
 of the patterns of cleavage within this sequence .
the majority of 
 cleavage sites were external to the common four base pair sequence 
 associated with tight feblm binding , suggesting a transient 
 scanning mechanism sufficient to allow double - strand cleavage at susceptible 
 sites external to the 5-acgc/5-gcgt sequence .
since 
 the binding of feblm to dnas 12 and 13 is consistent with a 1:1 affinity model , cleavage at sites distant 
 from the 5-acgc/5-gcgt sequence must logically involve 
 transient association with sites on the dna highly susceptible to 
 cleavage when they are populated .
this type of behavior is exemplified 
 by netropsin , which was demonstrated to undergo end - to - end flipping 
 without dissociating from a dna to which it was strongly bound .
it may be noted that for all four hairpin dnas 
 containing 5-acgc/5-gcgt ( dnas 2 , 7 , 12 , and 13 ) , the 5-acgc 
 sequence was always on the 5-arm of the hairpin and the 5-gcgt 
 sequence on the 3-arm .
most of the cleavage outside this common 
 tetranucleotide domain occurred toward the ends of the hairpin dna , 
 although the number of examples is insufficient to permit any firm 
 conclusion to be drawn as to the possible directionality of the putative 
 scanning process .
plausibly , the tight association of feblm 
 with these hairpin dnas as a consequence of the presence of the 5-acgc/5-gcgt 
 sequences may enable multiple double - strand cleavages of individual 
 dnas sufficient to render dna repair problematic .
the 30 hairpin 
 dnas isolated using immobilized fe(iii)blm 
 a5 for the selection procedure have sequences all of which 
 differ from the 10 hairpin dnas described to date .
it seems likely that some of these have sequences which are 
 analogous to 5-acgc/5-gcgt , in that their presence 
 is sufficient to confer strong blm binding properties to dnas in which 
 they are present .
further , the absence of sequence duplication within 
 any of the hairpin dnas isolated to date suggests that there must 
 be significantly larger numbers of sequences capable of mediating 
 strong binding to blm .
it seems possible that some of these could 
 prove to be more efficient than 5-acgc/5-gcgt in conferring 
 strong blm binding properties .
nonetheless , even in the absence 
 of such alternative tight binding 
 sequences it is worthy of note that the ncbi - blast program indicates that the 16-nt sequence 5-acgcacgcacgcacgc 
 occurs 32 times in the human genome at 
 sites within or close to protein coding regions of the genome ; these 
 could well constitute sites of enhanced cleavage by feblm .
in 
 fact , a 48-nt sequence consisting of 12 tandem repeats of acgc occurs 
 to the 5-side of gene b3gat1 ( gene i d 27087 ) on chromosome 
 11 which encodes glucuronosyltransferase p. other long tandem repeats 
 of acgc , or close variants thereof , occur within the genes for the 
 iq domain - containing protein g ( gene i d 84223 ) , cyclic amp - responsive 
 element - binding protein 5 ( gene i d 9586 ) , ribonuclease t2 precursor 
 ( gene i d 8635 ) , ferm domain - containing protein 4a ( gene i d 55691 ) , 
 and set - binding protein isoform ( gene i d 26040 ) .
this frequency of 
 occurrence for tandem repeats of acgc compared quite favorably with 
 that found for other similar but randomly chosen tetranucleotide sequences .
the study of feblm - mediated cleavage of strongly bound hairpin 
 dnas has already enabled a new mechanism of double strand dna cleavage 
 to be identified .
the present analysis 
 of hairpin dnas containing the tightly bound motif 5-acgc/5-gcgt 
 further suggests a mechanism by which multiple double - strand cleavage 
 reactions involving a single substrate dna could lead to the formation 
 of gaps in a dna structure , which would likely be quite difficult 
 to repair .
a further potential benefit to the identification of dna 
 motifs conducive to tight binding by feblm is related to the 
 likelihood that such motifs are involved in tumor cell killing by 
 bleomycin .
previous structural studies of blm  dna interaction 
 carried out by nmr and x - ray crystallographic 
 analysis have provided structural insights , 
 but inconsistency from one study to another , underscoring the need 
 for the use of an appropriate dna substrate .
the current findings 
 for the 5-acgc/5-gcgt motif suggest that dnas containing 
 this motif may constitute more relevant species for structural studies 
 of blm interaction .
a comparison of sequences 
 common among members of two hairpin dna 
 libraries revealed that the motif 5-acgc/5-gcgt was 
 present in four hairpin dnas , all of which bound feblm a5 exceptionally strongly . newly identified hairpin dnas 12 and 13 , containing the above motif , were characterized 
 in some detail .
dna 13 , which has a ka of about 30  10 m for fe(iii)blm b2 , binds the drug more tightly 
 than any dna previously identified . in keeping with the earlier finding 
 that dnas which bind feblm strongly exhibit enhanced double - strand 
 cleavage , and do so by a novel mechanism involving closely spaced 
 dna breaks , dna 12 was found to undergo five double - strand 
 dna breaks when treated with feblm a5 and four of 
 these occurred by the newly recognized mechanism . for dna 13 , all five double - strand breaks involved closely spaced single - strand 
 breaks .
it is interesting that tandem repeats of the motif 5-acgc/5-gcgt , 
 some up to 48 nucleotides in length , occur abundantly in the human 
 genome and may possibly constitute cellular targets for bleomycin .
[ -p]atp and [ -p]ddatp were purchased from 
 perkinelmer . dna polymerase ( klenow 
 fragment ) and restriction endonucleases asei , apoi and t4 polynucleotide kinase were obtained from new 
 england biolabs .
the 
 vector puc19 , plasmid mini kits , and competent cells dh5 were 
 from invitrogen .
fe(so4)2(nh4)26h2o was purchased from sigma - aldrich and 
 was used to prepare fresh aqueous solutions for admixture to blm a5 immediately prior to use .
chelex 100 was purchased from sigma - aldrich 
 and used to remove adventitious fe from solutions prior 
 to experiments .
oligonucleotides were purchased from integrated dna 
 technology , inc . the 41-nt template containing 8-nt random 
 sequences ( 2 g/l ) 
 was self - annealed in annealing buffer ( 10 mm sodium cacodylate , ph 
 7.0 , containing 100 mm nacl ) at 75 c for 15 min . the annealed 
 41-nt template ( 10 g ) was then treated with 12.5 l of 
 dna polymerase klenow fragment ( 62.5 u ) and 2.1 l of 10 mm 
 dntps in 10 mm tris - hcl , ph 7.9 , containing 50 mm nacl , 10 mm mgcl2 , and 1 mm dithiothreitol .
the reaction mixture was incubated 
 at 37 c for 30 min and then heated at 75 c for 20 min .
an aqueous 
 solution containing 2.0 mg ( 1.4 mol ) of blm a5 was 
 treated with 0.4 mg ( 1.4 mol ) of fecl36h2o , and the combined solution was stirred at room temperature 
 for 5 h and then lyophilized to afford a solid .
twenty - five 
 mg of sepharose 4b beads were added to 1.0 ml of 0.1 
 m sodium bicarbonate , ph 8.3 , containing 2.0 mg ( 1.4 mol ) of 
 fe(iii)blm a5 , and the resulting suspension was stirred 
 at 04 c .
after 36 h , the coupled fe(iii)blm a5sepharose 4b was washed with 4.0 ml of 0.1 m sodium bicarbonate , 
 ph 8.3 , and subsequently washed extensively with water to remove any 
 traces of free fe(iii)blm a5 .
the extent of bead 
 derivatization was determined from the supernatant buffer after the 
 coupling reaction was complete ; the absorption maximum at 292 nm is 
 characteristic for bleomycin , and the known molar absorptivity ( 14500 
 mcm ) permitted calculation 
 of the amount of bleomycin that had failed to undergo coupling .
resin bound fe(iii)blm a5 ( 2.0 
 nmol ) was incubated with 1.0 nmol of 64-nt hairpin dna in 20 l 
 ( total volume ) of 20 mm tris - hcl buffer , ph 7.4 , at room temperature 
 for 20 min .
the mixture was washed once with 20 l of 20 mm 
 tris - hcl buffer
. then the hairpin dna still bound to fe(iii)blm 
 a5 was isolated from the solid support by washing with 
 1 m nacl and desalted by amicon ultracentrifugal filtration ( millipore ) .
the 
 mixture of hairpin dnas eluted from resin bound fe(iii)blm a5 ( 250 ng ) was digested with 5 u of restriction endonuclease asei in 25 l of 50 mm tris - hcl , ph 7.9 , containing 
 100 mm nacl , 10 mm mgcl2 , and 1 mm dithiothreitol .
the 
 reaction mixture was incubated at 37 c for 1 h and then incubated 
 with 5 u of apoi at 50 c for 1 h. the enzymes 
 were inactivated by heating the solution at 80 c for 20 min .
the plasmid vector puc19 ( 4.5 g ) 
 was digested with 20 u of ndei and ecori - hf at 37 c for 3 h in 50 l of 20 mm tris - acetate , 
 ph 7.9 , containing 50 mm potassium acetate , 10 mm magnesium acetate , 
 and 1 mm dithiothreitol .
the incubation mixture was heated at 70 c 
 for 20 min to inactivate the enzymes .
the digested puc19 was isolated 
 from a 1% agarose gel and purified using a qia quick gel extraction 
 kit ( qiagen ) .
the asei / apoi digested 
 64-nt hairpin 
 dnas were incubated with the ndei / ecori digested plasmid puc19 in a 20 l reaction mixture having 
 5 u of t4 dna ligase in 50 mm tris - hcl , ph 7.5 , containing 10 mm mgcl2 , 10 mm dithiothreitol , and 1 mm atp .
the reaction mixture 
 was maintained at room temperature for 1 h. five l ( 21 
 ng ) of recombinant dna was added to 50 l of competent cells 
 dh5 preparation .
the incubation mixture was maintained on ice 
 for 30 min , followed by heating at 42 c for 20 s and cooling 
 on ice for 3 min .
the mixture was diluted with 1 ml of lb medium ( 10 
 mg / ml trypton , 5 mg / ml yeast extract , 10 mg / ml nacl , ph 7.4 ) and incubated 
 at 37 c with shaking at 150 rpm for 1 h. the cell suspension 
 was cultured on lb agar plates including 100 g / ml ampicillin , 
 30 g / ml x - gal , and 1 mm iptg at 37 c overnight .
the white 
 colonies were transferred to lb broth including ampicillin ( 100 g / ml ) 
 and incubated at 37 c overnight .
the isolation and purification 
 of recombinant plasmid dna were carried 
 out using invitrogen plasmid mini kits .
dna sequencing was carried 
 out in the dna laboratory of the school of life sciences at arizona 
 state university .
the sequences so determined enabled the identification 
 of the 64-nt hairpin dnas selected from the original library ; these 
 were prepared for study by chemical synthesis .
a solution formed 
 by admixture of 1.2 l of 30 m 64-nt 
 hairpin dna and 0.25 l of 144 m blm a5 was 
 pre - incubated for 20 min .
the solution containing the 64-nt hairpin 
 dna and blm a5 was added to a 16-nt hairpin dna - cf15 solution prepared by addition of 1.2 l of 30 m 
 hairpin dna - cf15 to 46.85 l of 10 mm na cacodylate 
 buffer , ph 7.0 , containing 100 mm nacl .
the reaction mixture was maintained 
 at room temperature for 1 min followed by the addition of 0.5 l 
 of 72 m freshly prepared aqueous fe(nh4)2(so4)26h2o .
the same volume 
 of buffer solution was added to the control sample without fe and 64-nt hairpin dna .
the final concentrations of 16-nt 
 hairpin dna - cf15 , 64-nt hairpin dna , and fe(ii)blm 
 a5 were all 0.72 m ( total volume 50 l ) .
the samples were 
 excited at 310 nm , and the emission signal was measured from 400550 
 nm using an excitation slit width of 10 nm and an emission slit width 
 of 10 nm .
hairpin 
 dnas were 5-end labeled using [ -p]-atp 
 + t4 polynucleotide kinase and 3-end labeled with [ -p]-atp + terminal deoxynucleotidyltransferase .
ten pmol of 
 64-nt hairpin dnas was [ 5-p]-end labeled by incubation 
 with 20 units of t4 polynucleotide kinase and 0.06 mci [ -p]-atp ( specific activity 6000 ci ( 222 tbq)/mmol ) in 50 l 
 ( total volume ) of 70 mm tris - hcl buffer , ph 7.6 , containing 10 mgcl2 and 5 mm dithiothreitol .
the reaction mixture was incubated 
 at 37 c for 1 h followed by purification of dna by 16% polyacrylamide 
 gel electrophoresis carried out at 2000 v for 2 h. the 3-end 
 labeling was done by incubating 10 pmol of hairpin dna with 20 units 
 of terminal deoxynucleotidyltransferase and 0.06 mci [  -p]-atp ( specific activity 6000 ci ( 222 tbq)/mmol ) in 50 l 
 ( total volume ) of 70 mm tris - hcl buffer , ph 7.6 , containing 10 mgcl2 , 10 mm cocl2 , and 5 mm dithiothreitol .
the reaction 
 mixture was incubated at 37 c for 1 h followed by dna purification 
 using 16% polyacrylamide gel electrophoresis carried out at 2000 v 
 for 2 h. febleomycin mediated cleavage of [ 5-p]- and [ 3-p]-end - labeled hairpin dnas 
 was carried out by incubating the hairpin dna ( 6000080000 
 cpm ) with 01.5 m fe(ii)bleomycin a5 at 25 c for 30 min in a solution of 5 l of 10 mm na 
 cacodylate buffer , ph 7.0 .
two l of native gel loading buffer 
 containing 0.25% bromophenol blue , 0.25% xylene cyanol , and 40% sucrose 
 were added to the bleomycin reaction mixture , which was resolved on 
 a 20% native polyacrylamide gel ( 260 v at 4 c for 20 h ) .
double - strand 
 cleavage sites were confirmed by visualizing gels using a phosphorimager 
 ( molecular dynamics ) .
co - migrating bands derived from an alternatively 
 [ 5-p]- and [ 3-p]-end - labeled 
 hairpin dna were presumed to arise from double - strand cleavage .
the [ 5-p]- and [ 3-p]-end - labeled 
 double - strand dna cleavage bands visualized by native gel electrophoresis 
 were extracted from the gel .
each gel slice was cut into several pieces , 
 placed in 0.3 ml of h2o , and incubated at 4 c overnight 
 to elute the dna , which was then concentrated under diminished pressure . 

each concentrated dna solution was admixed with 5 l of denaturing 
 loading buffer containing 80% formamide , 2 mm edta , 1% bromophenol 
 blue , and 1% xylene cyanol and heated at 90 c for 10 min .
five 
 l of the final solutions were chilled on ice and resolved in 
 a 16% denaturing polyacrylamide gel containing 7 m urea and run at 
 2000 v for 2.5 h. cleavage sites were confirmed by comparison with 
 the reaction products obtained through the maxam 
ten l of [ 5-p]- or 
 [ 3-p]-end - labeled dnas ( 20000 cpm ) recovered 
 from the native gel was treated with 25 l of formic acid and 
 incubated at 25 c for 45 min .
the reaction was stopped 
 by treatment with 200 l of 0.3 m naoac , ph 7.0 , containing 
 0.1 mm edta and 25 g / ml of trna . the resulting solution was 
 mixed with 700 l of ethanol , and the dna was precipitated . 

the dna pellet was washed twice with 70% ethanol , and the pellet was 
 resuspended in 75 l of 10% piperidine .
the reaction mixture 
 was incubated at 90 c for 30 min , and the cooled supernatant 
 was concentrated under diminished pressure .
the dna pellet was washed 
 with small amounts of water to remove residual piperidine and mixed 
 with denaturing loading buffer containing 80% formamide , 2 mm edta , 
 1% bromophenol blue , and 1% xylene cyanol .
the combined solution was 
 heated at 90 c for 10 min and applied to a sequencing gel to 
 compare [ 5-p]-end and [ 3-p]-end - labeled dnas by denaturing polyacrylamide gel electrophoresis .
spr measurements were performed with 
 a four - channel biacore t200 optical biosensor system .
flow cell 1 
 was left blank , while flow cells 24 were immobilized with 
 5-biotin - labeled dna sequences ( hairpin dnas 7 , 12 and 13 ) .
the spr experiments were performed at 15 and 25 c in degassed 
 and filtered cacodylate buffer ( 10 mm cacodylate , 0.1 mm edta , and 
 10 mm nacl , ph 7.2 ) .
solutions of different known fe(iii)blm 
 b2 concentrations were injected over the immobilized dna 
 surface at a flow rate of 75 l / min until a constant steady - state 
 response was obtained .
compound solution flow was then replaced by 
 buffer flow resulting in dissociation of the complex . after each cycle , 
 the sensor chip surface was regenerated with running buffer for 30 
 s followed by three buffer injections ( each 60 s ) to yield unbound 
 dna and a stable baseline for the following cycles .
the reference 
 response from the blank cell was subtracted from the response in each 
 flow cell containing dna to give a signal ( ru , response units ) that 
 is directly proportional to the amount of bound compound . the predicted 
 maximum response per bound compound in the steady - state region ( rumax )
was determined from the dna molecular weight , the amount 
 of dna on the flow cell , the compound molecular weight , and the refractive 
 index gradient ratio of the compound and dna , as previously described .
ru was plotted as a function of free ligand 
 concentration ( cfree ) , and the equilibrium 
 binding constants were determined with a one - site binding model ( k2 = 0).1where r represents the moles 
 of bound compound per mole of dna hairpin duplex , k1 and k2 are macroscopic binding 
 constants ( for a single - site model k2 = 
 0 ) , and cfree is the free compound concentration 
 in equilibrium with the complex .
rumax in the equation 
 was used as a fitting parameter , and the obtained value was compared 
 to the predicted maximal response per bound ligand to evaluate the 
 stoichiometry .
kinetic analysis was performed by globally fitting 
 the binding results for the entire concentration series using a standard 
 1:1 kinetic model with integrated mass transport - limited binding parameters 
 as described previously .
 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 feblm 
 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 feblm leads to 
 increased double - strand cleavage by a novel mechanism and identify 
 a specific dna motif conducive to strong blm binding and cleavage . 
Introduction
Results
Discussion
Conclusions
Experimental Methods