polymerases are critical 
 to the replication and repair of dna . while 
 replication of dna is an essential first 
 step for cell division , repair of dna
is needed when insults such 
 as uv rays , environmental toxins , and some drugs chemically modify 
 dna .
these modifications can yield a diverse 
 array of mutations . to understand the 
 mechanisms of dna replication and repair , it is crucial to understand 
 how a polymerase processes dna lesions . as part 
 of ongoing carcinogenesis research and to understand the 
 mechanisms of dna mutation and repair , we have been studying how the 
 bulky and mutagenic arylamine  dna lesions ( figure 1a ) interact with a polymerase or a repair protein . using f nmr , microcalorimetric , and other biophyisical 
 methods , we have shown that the arylamine lesions adopt three unique 
 conformations : base - displaced stacked ( s ) , major groove b - type ( b ) , 
 and minor - groove wedge ( w ) , depending on the location of the lesion 
 ( figure 1b ) .
the relative populations of s- , 
 b- , and w - conformers depend on the nature of attachment on the central 
 nitrogen ( n - acetyl vs n - deacetylated ) 
 and the hydrophobic carcinogen ring moiety ( planar vs twisted ) as 
 well as the base sequences ( flanking vs near long - range ) surrounding 
 the lesion .
( b ) major ( upper image ) and minor ( lower image ) groove views of the 
 prototype b- , s- , and w - conformers of arylamine  dg lesions 
 in cpk model with the dna duplex in gray surface ( color code : arylamine 
 lesion , red ; modified - dg , cyan ; dc opposite the lesion site , green ) . 
 note that the arylamine lesion ( red ) in w - conformation is wedged in 
 the narrow minor groove .
it has been shown that most replicative polymerases easily 
 bypass 
 the planar and n - deacetylated aminofluorene ( af ) 
 adducts after a brief stall at the lesion site .
on the other hand , 
 the bulkier n-(2-deoxyguanosin-8-yl)-2-acetylaminofluorene 
 ( aaf ) analogues can not be readily bypassed and thus stall dna synthesis . in in vitro studies with x - family 
 polymerase  , aaf adducts lead to 2 base deletion mutations , 
 while af extends full - length primers .
a recent study via single - molecule fluorescence spectroscopy showed 
 that high - fidelity polymerases can not extend a primer whose terminus 
 occurs across from aaf . in e. coli ,
aaf adducts result mostly in frameshift 
 mutations , while both af and aaf adducts cause point mutations . in mammals ,
this difference in mutagenic profiles has been 
 attributed to the presence of a bulky acetyl group on the central 
 nitrogen , which causes the aaf adduct to adopt a syn conformation .
in contrast , the af adduct 
 adopts an anti-/syn - conformation , 
 while the n-(2-deoxyguanosin-8-yl)-4-fluoro-4-aminobiphenyl 
 ( fabp ) adduct adopts exclusively an anti - conformation .
other factors influencing adduct - induced mutations 
 include topology , insertion of the nucleotide opposite the lesion 
 site , and the characteristics of the polymerase . numerous crystal structure and kinetic analysis studies are 
 available 
 and provide information on actions of native and damaged dna with various polymerases .
however , only few examples of replicative 
 polymerases complexed with bulky arylamine - modified dna are available 
 with atomic resolution details , presumably due to difficulties 
 with obtaining crystals .
high - resolution solution nmr can offer dynamic 
 information alternative to that of static crystallography .
however , some bulky dna lesions cause conformational variation in 
 the dna that can also occur upon binding with a polymerase , which 
 introduces additional challenges to the use of this method . as a result , 
 most nmr studies thus far are limited to adducted dna without the 
 full presence of polymerases and repair proteins . theoretical / molecular dynamic simulations in conjunction with limited 
 nmr and crystal data have been useful .
other available 
 techniques for biomolecular interactions such as 
 electrophoretic mobility ( gel shift or gel retardation assay ) and 
 filter - binding assays provide valuable information on binding affinity . 
 however , these approaches contribute either little or no insight on 
 the kinetic parameters underlying complex formation . moreover , these 
 techniques require strenuous work to determine binding parameters .
in addition , gel assays do not allow the samples 
 of interest to be in chemical equilibrium due to fast dissociation 
 rate during electrophoresis ; thus , it is difficult to measure proper 
 binding kinetics and thermodynamics . finally , 
 microcalorimetry , such as isothermal titration calorimetry ( itc ) , 
 is a fast and robust method that certainly could be used to characterize 
 binding interactions and the thermodynamics of polymerase dna interactions 
 in free solution , but low - affinity interactions would require higher 
 protein concentrations .
surface 
 plasmon resonance ( spr ) is a powerful , chip - based , and 
 label - free solution technology that can provide real - time information 
 on kinetics and thermodynamics .
spr relies on changes in the refractive index that are due to changes 
 in mass and can thus measure a small difference in binding ( kd ) at the subnanomolar level .
we have recently communicated our initial spr work on 
 the binding affinities of kf - exo to arylamine dna 
 lesions .
subsequently , a similar study was conducted 
 to elucidate how faf lesions affect the active site conformation of 
 the human repair enzyme pol  and how the structure and sequence 
 of the dna affects its ability to be repaired . in the present study
, we are providing a complete set of spr 
 data 
 on the binding of kf - exo or pol  to faaf 
 and fabp lesions in two different sequences ( cg*a and tg*a ) . to complement 
 the spr binding results
, we also conducted dynamic f 
 nmr as well as steady - state nucleotide insertion kinetics .
the results 
 are discussed in terms of adduct - induced conformational heterogeneity , 
 the effect of the 5-flanking base sequence , substrate specificity , 
 and the nature of a polymerase .
the purpose of the present article 
 is 2-fold : ( 1 ) to give the full details of our previous spr work and ( 2 ) to introduce spr to the chemical toxicology 
 community as a powerful alternative to existing techniques for investigating 
 protein  dna interactions . as a result ,
the choice of polymerases 
 used in the present study was based largely on the experimental systems 
 in our previous work .
obviously , 
 future spr studies should be expanded to a range of y - family bypass 
 polymerases , which are more likely to be involved in replication of 
 bulky dna lesions .
dna sequences 
 containing 5-biotin - labeled 31-mer oligonucleotides , 
 phosphorylated 52-mer hairpin , and 21-mer complementary sequences 
 ( figure 2b , c ) were purchased from operon ( eurofin , 
 huntsville , al ) in desalted form and purified by reverse - phase high - performance 
 liquid chromatography ( rp - hplc ) .
the hplc system 
 consisted of a hitachi ezchrom elite hplc system with an l2450 diode 
 array detector and a clarity column ( 10 mm  150 mm , 3 m ) 
 ( phenomenex , torrance , ca ) .
the mobile phase system involved a 20 
 min linear gradient profile from 3 to 16% ( v / v ) acetonitrile with 
 100 mm ammonium acetate buffer ( ph 6.5 ) at a flow rate of 2.0 ml / min . 

kf - exo ( d424a ) and pol  were received as 
 gifts from dr .
catherine joyce ( yale university , new haven , ct ) and 
 dr . william beard ( niehs , research triangle park , nc ) .
primer oligonucleotide constructs 
 for ( b ) kf - exo and ( c ) pol . approximately 70 m of a faaf 
 or fabp  dg modified 16-mer template was annealed with a 9-mer 
 primer in a 1:1 molar ratio to produce a ds / ss junction containing 
 duplexes ( figure 3 ) .
the samples were lyophilized 
 and dissolved in 300 l of typical ph 7.0 nmr buffer containing 
 10% d2o/90% h2o with 100 mm nacl , 10 mm sodium 
 phosphate , and 100 m edta .
all f nmr spectra were 
 recorded using a dedicated 5 mm f / h dual 
 probe on a varian 500 mhz spectrometer operating at 476.5 mhz , using 
 acquisition parameters described previously .
the spectra were acquired in the h - decoupled mode and referenced relative to that of cfcl3 by assigning external c6f6 in c6d6 at 164.9 ppm .
f nmr spectra were 
 measured at two different temperatures , 5 and 25 c .
single - nucleotide / full - length 
 extension experiments for both fabp and faaf  dg adducts 
 in kf - exo were performed as described previously .
briefly , the 9-mer primer was 5-radiolabeled 
 using [ -p]atp and t4 polynucleotide kinase ( t4 
 pnk ) following the manufacturer s protocol .
the p - labeled primer ( 50 pmol ) was annealed with either an unmodified 
 or adducted template oligonucleotide ( 60 pmol ) by heating at 95 c 
 for 5 min and then slowly cooling to room temperature in 3 h. for 
 pol  assays , a 1 nt gap was generated by adding a downstream 
 9-mer primer with 5-phosphate group while annealing with radiolabeled 
 primer ( 9-mer ) and template ( 19-mer ) .
the 
 ds / ss primer  template sequence ( 20 nm ) was incubated with kf - exo ( 0.5 or 1.0 nm ) for 5 min to form a binary complex 
 in tris buffer ( tris , 50 mm , ph 7.4 ; bsa , 50 g / ml ; 5% ( v / v ) 
 glycerol ) .
the reaction was initiated by adding a dntp ( 100 m)/mgcl2 ( 5 mm ) solution to a binary mixture and was incubated at 
 22 c for 10 min .
the reaction was arrested with gel loading 
 buffer ( containing 50 mm edta ( ph 8.0)/95% formamide solution ) .
the 
 quenched sample was heated at 95 c for 5 min and immediately 
 cooled on ice .
the products were resolved with a denaturing polyacrylamide 
 gel ( 20% polyacryamide ( w / v)/7 m urea ) , electrophoresed at 2500 v 
 for 4 h. the gel was exposed on a kodak phosphor imaging screen overnight 
 and scanned with a typhoon 9410 variable mode imager . to determine the efficiency 
 of dctp insertion opposite the adducted site ,
steady - state kinetic 
 parameters for incorporation of the nucleotide opposite the unmodified 
 and fabp - modified templates were determined by using the reported 
 literature procedures .
the reactions were performed with 
 pol  ( 0.5 nm ) and oligonucleotide ( 20 nm ) at 22 c . for 
 the unmodified sequence
, reactions were performed in a shorter time 
 period of 0.510 min for nucleotide incorporation and up to 
 30 min in the case of modified templates .
the percentage 
 of primer extended in kinetic assays was determined by taking the 
 ratio of extended primer to the total amount of primer ( unextended 
 + extended primer ) .
the modification of 5-biotin 
 cga / tga sequences ( 31-mer ) was 
 carried out using the previously reported procedures ( figure 2 ) , and the modified products were 
 purified by rp - hplc and characterized by maldi - tof mass spectrometry . 

biotinylated unmodified ( 20 m ) or modified 31-mer ( 20 m ) 
 was annealed with 20 m of 52-mer hairpin by heating at 95 c 
 for 5 min and cooling to room temperature ( figure 2 ) .
the annealed mixture was ligated by using 4000 u t4 dna 
 ligase in 1 ligase buffer for 16 h at room temperature .
the 
 ligated 83-mer oligonucleotide was purified by 10% denaturing polyacrylamide 
 gel ( figure s1 ) and extracted using the 
 crush and soak method .
the desalted oligonucleotide was 
 incubated with 2,3-dideoxy - thymidine-5-triphosphate 
 ( ddttp ) ( 1 mm ) in the presence of kf - exo ( 1 m ) 
 and 5 mm mgcl2 for 12 h. the dideoxy - terminus dna was purified 
 by rp - hplc ( figure s2 ) after precipitation 
 of protein using phenol 
either 
 a biotinylated 31- , 83- , or 84-mer dna sequence ( 100 pmol ) was mixed 
 with 2 l of matrix containing 1 l of 3-hydroxy picolinic 
 acid ( 3-hpa ) ( 50 mg / ml dissolved in acetonitrile / water 50% v / v ) and 
 1 l of diammonium hydrogen citrate ( dahc ) ( 50 mg / ml dissolved 
 in acetonitrile / water 50% v / v ) .
the mass spectrometric 
 measurement of 31-mer oligonucleotides was carried out in a reflectron 
 positive mode .
the calibration of the instrument in reflectron positive 
 mode was performed using low - molecular - weight oligonucleotide or peptide 
 standard calibration kit . for high - molecular - weight oligonucleotides 
 ( > 10  000 da ) , calibration was done in a linear negative 
 mode 
 using 52- , 80- , 90- , and 100-mer standards with laser power 120 in 
 order to enhance the signal intensity .
the spectral data was processed 
 by using shimadzu biotech maldi - ms software with processing parameters 
 as follows : smoothing filter width as 20 channels , baseline filter 
 width as 80 channels , and double threshold .
a carboxymethylated dextran - coated 
 cm5 chip supplied by ge healthcare was used to immobilize streptavidin 
 ( sa ) via the amine coupling kit on flow cells by following the previously 
 reported literature .
the 
 edc / nhs mixture was injected over the surface for 7 min followed by 
 sa ( 50 g / ml dissolved in sodium acetate buffer , ph 4.5 ) .
the 
 unreacted reactive esters were blocked with 1 m ethanolamine for 7 
 min .
the running buffer used for immobilization was 1 hbs - ep buffer containing 10 mm hepes ( ph 7.4 ) , 150 mm nacl , 3 mm 
 edta , and 0.05% nonionic surfactant p20 .
after sa immobilization , the surface was washed 
 with 50 mm naoh for 60 s pulse , which was repeated five times to remove 
 the free sa until the change in response unit reached below 20 ru . 

the surface was stabilized by injecting ( 34 times ) running 
 buffer followed by equilibration with running buffer for 1 h. the 
 84-mer biotinylated dna - hairpin sequences of either unmodified or 
 adducted dna ( 0.250.3 nm ) were injected over the flow cells 
 2 or 4 for 60120 s individually to achieve 0.73.5 
 ru .
the flow cells were washed with running buffer to remove the unbound 
 dna and to stabilize the surface . before conducting kinetics experiments , 

1 mm ddttp in the presence of kf - exo ( 1 m ) 
 and 5 mm mgcl2 was injected over the surface for 5 min 
 followed by 0.05% sds to remove the polymerase . for pol  experiments , 

a 1 nt gap was created by using the same dna coating approach , and , 
 in addition , a corresponding downstream complementary sequence ( 21-mer ) 
 containing a 5-phosphate group ( 2 nm ) was injected over the 
 surface for 5 min .
kf - exo was injected with or without dntps ( 100 m ) 
 over the dna surface 
 in random order ( neither ascending nor descending concentrations ) . 

each concentration was repeated twice . for the binary system , varying 
 concentrations of kf - exo ( 010 nm ) prepared 
 in running buffer containing 1 hbs - p along with 
 100 g / ml bovine serum albumin ( bsa ) and 5 mm mgcl2 was used .
the polymerase was injected for 30 s at a flow rate of 
 100 l / min followed by dissociation of polymerase .
the surface 
 was regenerated using 0.05% sds at a flow rate of 100 l / min , 
 and the injection time was 30 s followed by an extra wash with running 
 buffer .
after regeneration of the surface , the surface was stabilized 
 with running buffer for 15 min .
initially , three startup steps with 
 running buffer and four zero - concentration injections were performed 
 to condition the surface . for the ternary system ,
individual dntp 
 ( 100 m ) was mixed with varying concentrations of kf - exo and injected over the surface .
the binding 
 affinity constants ( kd ) for binary and 
 ternary systems were calculated using steady - state affinity analysis 
 in biaevaluation software v1.0 as the association rate for the ternary 
 system , particularly as dctp and unmodified dg are near the diffusion 
 limit .
the sensorgrams for binary systems were globally fitted with 
 biasimulation basic kinetics module software by using experimental ka and kd values 
 ( figure s7 ) .
similar experiments 
 were carried out for pol  interaction studies with adduct present 
 both at the nongapped duplex dna and 1 nt gap dna .
single - nucleotide 
 ( 1 nt)-gapped dna was generated by annealing a corresponding downstream 
 primer . for nongapped dna , the concentration of pol  was varied 
 up to 1000 nm , whereas for 1 nt gap , it was 0100 nm depending 
 on the dg adduct embedded .
an overall 
 scheme for the construction of the biotinylated hairpin - based template  primer 
 strands is depicted in figure 2a .
fabp- or 
 faaf - modified biotin-31-mer oligonucleotides were prepared according 
 to published procedures .
the 52-mer hairpin dna was annealed and ligated to the biotinylated 
 31-mer ( figure 2b , c ) .
the hairpin structure was created to improve 
 the thermal stability of the oligonucleotide constructs on a gold 
 chip during kinetics experiments . as a result
, the same oligonucleotide 
 constructs could be used multiple times with different polymerases 
 and buffer conditions .
finally , the lesion was positioned at the 22nd 
 base , with 21 bases on the 5-side and 28 bases on the 3-side , 
 in order to avoid close contact between the polymerase and the chip 
 surface .
the resulting template / primer strands , containing the biotinylated 
 84-mer hairpin , were purified by denaturing polyacrylamide gel ( figure s1 ) and used for further study .
maldi - tof spectrum , obtained in reflectron mode , of the faaf - modified 
 biotin - tg*a-31-mer sequence is shown in figure 
 s3 .
a distinctive peak at 9841.30 da is in close agreement 
 with theory ( 9839.90 da , m / z : + 1.40 ) , and the inset is a linear negative mode spectrum .
the inset 
 b at 25925.76 da corresponds to the 83-mer strand consisting of the 
 biotin-31-mer - tg[faaf]a- and the 52-mer hairpin in the absence of 
 ddt at the primer terminus ( theoretical 25923.00 da , m / z : + 2.76 ) .
the inset c at 26206.70 da 
 corresponds to the 84-mer strand formed by adding ddt to the primer 
 terminus of the 83-mer strand ( 26211.00 da , m / z : 4.30 ) .
the corresponding tg[fabp]a , 
 cg[faaf]a , and cg[fabp]a sequences were similarly characterized ( figure s4s6 ) .
all of the calculated and 
 experimental m / z values are shown 
 in table s1 . to examine lesion - induced conformational 
 heterogeneity , we measured f nmr spectra of modified 
 16/9-mer template / primer duplexes .
as shown in figure 3 , the f 
 nmr spectra of fabp- and faaf - modified duplexes in the cg*a and tg*a 
 sequences are compared at 25 c .
fabp duplexes exhibited a single 
 peak at 116.4 ppm in both sequences , which is consistent with 
 the chemical shift range observed previously for the anti - b - type fabp conformer .
the bulky faaf 
 displayed three f signals , with two prominent peaks of 
 similar intensity at around 114 to 116 ppm , for both 
 sequences .
we have previously reported the chemical shift ranges that 
 correspond to the b- , s- , and w - conformers of faaf - modified duplexes , 
 i.e. , 115.0 to 115.5 ppm for the b - conformer , 115.5 
 to 117.0 ppm for the s - conformer , and 117.0 to 118.0 
 ppm for the w - conformer .
hence , the present 
 faaf - induced heterogeneity could be a variation of the b / s / w heterogeneity . 

in contrast to the aforementioned study , however , the f signals in the present study are derived from the lesions at the 
 ds / ss junction , not fully paired double helical duplexes .
the relative shielding of f signals and the narrow 
 chemical shift range ( 2 ppm ) in the present work are probably 
 due to the flexible lesions at the ds / ss junction . as a result
, we 
 could not unequivocally assign the signals to the b- , s- , or w - conformer .
f nmr spectra of fabp and faaf adducts in the cga 
 and tga duplexes at a ds / ss junction at 25 c .
single - nucleotide 
 incorporation 
 was carried out using the e. coli exonuclease - deficient 
 klenow fragment ( kf - exo ) and the human base excision 
 repair polymerase  ( pol  ) ( figure 4 ) . like any other high - fidelity replicative polymerase
, kf - exo prefers the ds / ss replication fork as a template / primer 
 dna substrate . in the unmodified dna control ,
the primer was immediately 
 elongated to full length in the presence of all four nucleotides and 
 kf - exo ( data not shown ) . with the fabp - modified 
 template ,
however , primer elongation was largely stalled at the lesion 
 site , with some insertion of the correct dctp opposite the lesion 
 ( figure 4a ) .
assays of full - length and single - nucleotide 
 incorporation into 
 fabp - adducted cg*a and tg*a sequences with ( a ) kf - exo and ( b ) pol . unlike kf - exo , pol  prefers a single - nucleotide 
 gap as a substrate . with pol  , there was no 
 full extension of either the unmodified ( not shown ) or fabp - modified 
 template ( figure 4b ) .
as for faaf , no nucleotide 
 insertion was observed with either kf - exo or pol 
  , even at high enzyme concentrations or with a longer incubation 
 period ( data not shown ) , because the lesion had completely blocked 
 elongation .
we conducted 
 steady - state experiments 
 to investigate the impact of conformational heterogeneity on nucleotide 
 insertion kinetics .
the results for kf - exo and 
 pol  are summarized in tables 1 and 2 , respectively . to examine the influence of lesions
, 
 we used the relative insertion efficiency fins , which was defined as ( kcat / km)modified  or  mismatched /(kcat / km)unmodified . with kf - exo , the fins of dctp opposite -cg[fabp]a- was 500-fold 
 lower than that of the unmodified control ( table 1 )
this is contrasted with -tg[fabp]a- , which was reduced 
 only 33-fold . in the pol  assay ( table 2 ) , the fins of dctp opposite fabp in 
 the cga sequence was 142-fold lower than that of the control , while 
 in the tga sequence , the fins was 59-fold 
 lower than that of the control .
these results indicate that the nucleotide 
 insertion efficiency is consistently greater in the tga sequence compared 
 to that in the cga sequence , regardless of the polymerase structure . 

we were unable to perform similar steady - state kinetics experiments 
 for faaf because this lesion caused a major blockage at the replication 
 fork .
after 
 activation with streptavidin ( sa ) , flow cells 1 and 3 were retained 
 as blank references , and dna was coated on the sa surface of flow 
 cells 2 and 4 .
surface testing , regeneration buffer scouting , and 
 the mass transport limitation test were performed before the kinetics 
 experiments , as described previously .
dna 
 coating at 0.7 resonance units ( ru ) did not show any influence of 
 mass transport ; an increase in flow rate of the analyte did not alter 
 the association rate .
however , at 10 ru , mass transport became a limiting 
 factor , as the association rate deviated with the flow rate of the 
 analyte ( data not shown ) .
on the basis of this study of mass transport 
 limitation , all of the experiments were carried out in the dna coating 
 range between 0.7 and 3.5 ru .
the sensorgrams for the binary 
 binding between kf - exo and the unmodified tga controls 
 or the modified tg*a oligonucleotide constructs are shown in figure 5a .
we performed steady - state affinity analysis of 
 the binary and ternary complexes in the presence of four dntps ( figure 6 ) .
a similar set of results for the cga sequence 
 have been reported previously , and the 
 results on the binding affinity of kf - exo to both 
 tga and cga sequences are summarized in table 3 .
sensorgrams of binary complexes of ( a ) kf - exo and 
 ( b ) pol  with unmodified and modified tga sequences ( 1:1 
 binding fitted curves are overlaid as red lines ) .
steady - state affinity analysis of interaction of kf - exo with ( a ) -tg[faaf]a- and ( b ) -tg[fabp]a- sequences .
as for the unmodified 
 controls , kf - exo binds 
 tightly in both sequences in the presence of the correct dctp .
the 
 affinity of binding for the cga sequence was reduced by 30- , 62- , 
 264- , and 34-fold in binary , datp , dgtp , and dttp , respectively , compared 
 to that for the correct dctp binding ( table 3 ) .
similar results were obtained for tga , where the binding affinity 
 was reduced by 15- , 39- , 180- , and 40-fold in binary , datp , dgtp , 
 and dttp , respectively ( table 3 ) .
these results 
 are consistent with those of the nucleotide insertion assay , which 
 showed preferential insertion of the correct dctp .
kf - exo bound strongly to the modified tg*a templates . 
 in the tg*a sequence ,
the kd value for 
 fabp was 4.9-fold greater than that of the control , and the kd value for faaf was 8.8-fold greater than that 
 of the control .
similar changes were observed in the cg*a sequence , 
 where the kd for fabp was 10.8-fold larger 
 than that for the control , and the kd for 
 faaf was 7.2-fold larger than that for the control .
these differences 
 are primarily due to the much slower dissociation rates observed for 
 the modified template / primer for both the cg*a sequence ( faaf kd : 0.02 s ; fabp kd : 0.01 s ) and the tg*a 
 sequence ( faaf kd : 0.01 s ; fabp kd : 0.01 s ) .
the net stabilization energies were positive and ranged from 1.10 
 to 1.47 kcal / mol ( table s2 ) .
kd for the correct nucleotide was 0.190.25 
 nm with faaf and 0.290.30 nm with fabp , while for the incorrect 
 nucleotide , kd was 0.280.67 nm 
 with faaf and 0.310.66 nm with fabp . for 
 pol  , binding assays were performed 
 on two distinct substrates : nongapped ds / ss and 1 nt gap .
the results 
 for the binary and ternary systems on both cga and tga sequences are 
 summarized in table 4 .
weak binding was observed 
 for the nongapped dna , with kd values 
 of 0.8 m ( data not shown ) .
in contrast , the binding 
 affinity of pol  increased 1000-fold with the 1 nt gap .
kd values 
 are in nanomolar ( nm ) . for 
 nongapped duplex dna ( ds - dna ) 
 with pol  , kd values exceeds 0.8 
 m . as for the unmodified 
 controls , pol  binds to the correct 
 dctp more tightly .
the binding affinity for the dctp is 2.7-fold higher 
 in the tga sequence and 4.5-fold higher in the cga sequence ( table 4 ) .
in contrast to kf - exo , the 
 binding in the binary complex between the modified template and pol 
  is less tight than that in the complex containing the unmodified 
 template , where the differences in binding are approximately 3-fold 
 for fabp and 5- to 6-fold for faaf , respectively .
similar to kf - exo , the binary complex with faaf showed slower off rates 
 ( kd : 0.01 s ) with 
 pol  in both sequences .
faaf ( figure 5b ) are relatively poor ; however , the residual plots 
 for the dg  faaf / pol  binary complex ( figure s10 ) indicate a good curve fit within 1%  values of rmax . the complexes 
 with b - conformeric fabp exhibited unusually faster dissociation rates 
 for both the cg*a and tg*a sequences , where the kd values were 0.76 and 0.40 s , respectively , 
 and the negative net stabilization energy was 1.04 and 0.27 
 kcal / mol , respectively ( table s2 ) .
figure s9 shows the sensorgrams for 
 the ternary complexes between pol  and the faaf- and fabp - modified 
 cg*a constructs .
we have recently reported a similar set of binding 
 results for the n - deacetylated faf . with the correct nucleotide dctp ,
the pol  binds 
 2.7-fold more tightly in the ternary complex than that in the binary 
 complex and 3000-fold more tightly than to that of nongapped 
 dna .
the binding affinity to the incorrect nucleotide was 4- to 5-fold 
 lower than that to the correct dctp .
the lesion in the 1 nt gap reduced 
 the binding affinity of pol  by 6-fold for faaf and 3-fold 
 for fabp , virtually eliminating the nucleotide selectivity of pol 
  at the lesion site .
the affinity for pol  binding decreased 
 in the order dg > fabp > faaf .
in 
 the present study , we have employed spr to investigate the binary 
 and ternary binding interactions of kf - exo and 
 pol  to two prototype arylamine  dna lesions ( fabp and 
 faaf ) in the context of two different sequences ( cg*a and tg*a ) .
kf - exo is a 68 kda high - fidelity replicative a - family bacterial 
 dna polymerase , which carries polymerase 
 and 35 exonuclease activities and has been 
 used extensively as a model enzyme for studying adduct - induced dna 
 synthesis .
pol  is the smallest ( 39 kda ) eukaryotic polymerase , 
 belonging to the x - family of base excision repair dna polymerases , 
 and has been characterized extensively . with pol  ,
primer extension past aaf adduct was blocked , 
 but full - length products were shown to contain exclusively 2 
 deletion mutations . although its role 
 is limited in base excision repair , pol  has been additionally 
 implicated in the replication of various dna damage .
for example , 
 deregulation of pol  may enhance the genetic instability induced 
 by bulky lesions such as cisplatin and 
 uv radiation .
fabp and faaf are c8-substituted 
 dg adducts that contain structurally unique arylamine structures , 
 i.e. , n - acetylated / coplanar - fluorene and n - deacetylated / twisted - biphenyl , respectively ( figure 1a ) . finally , the two sequences ( cg*a vs tg*a ) were selected because of their 
 marked difference in the s / b population ratios observed with the n - deacetylated faf .
the spr 
 results , along with data from f nmr and steady - state 
 primer kinetics , elucidate how lesion - induced conformational heterogeneity 
 alters the binding capacity of a polymerase and thus its nucleotide 
 insertion efficiency . we constructed the 84-mer hairpin - based 
 oligonucleotides for spr 
 ( figure 2 ) based on the following considerations . 

first , the incorporation of ddt at the 3-end of the primer 
 prevents the usual nucleophilic attack of the 3-hydroxyl to 
 the incoming dntp and thus blocks the formation of a phosphodiester 
 bond .
this ensures the stability of the ternary 
 complex polymerase / template - primer / dntp for spr measurements .
previous 
 assays using gel electrophoresis , single - molecule fret , or crystallography 
 have consistently shown that the absence of 3-oh at the primer 
 terminus does not affect the affinity with which polymerases bind 
 to binary and ternary complexes of dna .
second , while 
 kf - exo requires a minimum of 11 bases , because 
 it covers approximately 5 bases downstream from the primer / template 
 junction and 6 to 7 bases upstream of the 3-primer s 
 terminus , pol  can operate on 
 any length of dna containing a 1 nt gap .
we observed very tight binding of kf - exo with native unmodified dg in the presence of the correct incoming 
 nucleotide dctp .
this system exhibited high nucleotide selectivity , 
 with kd values increasing in the order 
 dctp  dttp  datp  dgtp ( table 3 ) .
the spr results are in agreement with nucleotide insertion 
 assays , which showed exclusive insertion of the correct dctp over 
 other dntps .
2:1 and higher order complexes have also been observed 
 in solution by various biochemical and biophysical methods .
as shown in figure s11 , comparison between 
 theoretical and experimental rmax for 
 pol  and kf - exo are in good agreement , indicating 
 a 1:1 complex .
initially , we carried out a spr binding assay 
 of pol  using the nongapped ds / ss junction replication fork . 

the binding was very weak , with kd values 
 in the micromolar concentration range .
however , upon introduction of the 1 nt gap ( figure 2c ) , the dna binding affinity of pol  increased 200- 
 to 1000-fold .
these results indicate that the presence of 5-po4 enhances the binding affinity of the 8 kda lyase domain as 
 well as the 31 kda catalytic domain .
the observed differences in binding 
 affinity are consistent with previous reports in which the lyase domain 
 in the duplex ( nongapped ) dna was flexible .
introduction of the 1 
 nt gap enhances the binding affinity of the polymerase to dna .
the results are also in agreement with gel assays , 
 which have previously shown that addition of the correct dctp opposite 
 unmodified dna enhances the binding affinity of polymerase compared 
 to that with other nucleotides by an induced - fit model adopted by 
 pol . an unusually greater binding of kf - exo was observed for modified dg , where the kd of this interaction was 511-fold higher than the kd for interaction with the unmodified native 
 dna substrate .
the binary binding affinity decreased in the order 
 fabp > faaf > dg for the cg*a sequence and faaf > fabp > 
 dg for the 
 tg*a sequence ( table 3 ) .
previous studies have 
 also shown tighter binary binding of kf - exo with 
 the aaf adduct . using gel - retardation 
 assays , dzantiev and romano showed that 
 the bulky and hydrophobic aaf interacts with nearby hydrophobic amino 
 acid residues , strengthening its binding to the active site of kf - exo .
the authors suggested that such lesion - induced conformational 
 adjustment may block the conformational change required to properly 
 accommodate an incoming nucleotide .
it is well - established that the n - deacetylated fluorinated 
 analogue faf adducts ( figure 1 ) adopt sequence - dependent 
 equilibrium between b- and s - conformers .
fabp is similarly n - deacetylated , but it lacks a methylene bridge , resulting 
 in a bulky twisted biphenyl moiety . in 
 other words , fabp may behave like faaf at the replication fork of 
 the template in the active site of a polymerase .
in contrast to the 
 unmodified control , modified adducts displayed a significant decrease 
 ( 713-fold ) in dissociation rate , with positive net stabilization 
 energy ( table s2 ) .
the markedly slower 
 off rates are consistent with single - molecule fret studies as well 
 as gel shift assays in which the presence of the bulky dna adduct 
 stabilizes the binary complex and does not induce dissociation before 
 nucleotide incorporation .
in contrast to 
 kf - exo , pol  exhibited 
 significantly less binary binding affinity to the modified templates . 

furthermore , the modified sequences exhibited significantly faster 
 dissociation rates and more negative net stabilization energies .
as 
 in the ds / ss situation discussed above , it is likely that faaf promotes 
 conformational heterogeneity in a sequence containing a 1 nt gap . 

such heterogeneity may hinder the interaction of that sequence with 
 key amino acids in the polymerase , thus preventing the polymerase 
 from undergoing conformational change that is necessary for strong 
 binding .
nucleotide selectivity was low in the ternary 
 complexes with kf - exo , where the kd values indicate poor discrimination between the correct 
 ( kd 0.190.30 nm ) and incorrect 
 ( kd 0.280.67 nm ) nucleotides . 

this poor selectivity does not depend on the 
 nature of the lesion ( fabp vs faaf ) or the 5-flanking base 
 ( cg*a vs tg*a ) .
the lack of nucleotide selectivity appears 
 to be in agreement with the results of tryptic digestion studies , 
 in which the aaf  polymerase complex maintains an unstable noncatalytic 
 open conformation in the presence of any dntp .
in other words , aaf modification did not stabilize the 
 complexes in relation to the incoming nucleotide .
this is contrasted 
 with native dna , to which the polymerase binds very tightly in the 
 presence of the correct nucleotide dctp and is insensitive to digestion . 

our f nmr results ( figure 3 ) indicate 
 a complex conformational heterogeneity of the bulky faaf at the ds / ss 
 templating position , which may prevent the polymerase from properly 
 accommodating an incoming dntp .
this reasoning is in accord with the 
 weak electron densities observed for the arylamine base in the active 
 site of t7 dna polymerase , where the 
 authors of the previous study also concluded that conformational heterogeneity 
 may hinder the insertion of an incoming nucleotide .
the low 
 selectivity for incoming nucleotides could also arise from the high 
 stability of the binary complex , which may hinder the polymerase s 
 ability to recognize the incoming nucleotide .
no crystal structures 
 or high - resolution nmr structures are currently available for complexes 
 between any dna polymerase and abp or the fluorinated fabp . in the 
 present study , fabp in both sequences exhibited a single f signal , possibly for a b- or a b / s - conformational mix , owing to 
 the presumed conformational flexibility at the ds / ss junction .
these 
 nmr data , albeit in the absence of a polymerase , are in agreement 
 with the gel - based kinetics data , which reveal a preference toward 
 inserting the correct nucleotide over other nucleotides ( table 1 ) . in the case of kf - exo ,
the tg*a sequence favored 
 the insertion of dctp more efficiently than the cg*a sequence .
the 
 relative insertion efficiency fins of 
 dctp opposite fabp was significantly lower in the cg*a ( 500-fold ) 
 and tg*a ( 33-fold ) sequences compared to that of the unmodified controls 
 ( table 1 ) .
this 15-fold difference in fins is puzzling because fabp at the ds / ss junction 
 exhibited a single f signal in both sequences ( figure 3 ) .
however , we have shown previously that faf in 
 the duplex setting displayed a greater s - conformer in the cg*a duplex 
 ( 50% ) relative to that in tg*a ( 38% ) . as mentioned above
, it is likely 
 that the absence of coplanarity in fabp would embrace intermediate 
 structures between faaf and faf , as observed from f nmr , 
 gel , and spr assays .
the spr results with pol  ( table 4 ) indicated that a modified templating base weakens 
 the polymerase s 
 binding affinity and the nucleotide selectivity ( figure s9 , table 4 ) .
the reduced binding 
 affinity of pol  to the modified template dna could be related 
 to the lesion - induced conformational heterogeneity in the active site 
 of the polymerase . in the closed conformation ,
key amino acids such 
 as lys 234 and tyr 271 interact with the minor groove of the primer 
 strand , while arg 283 interacts with the template strand of dna . as 
 mentioned above , it is possible that the faaf at the 1 nt gap may 
 hinder the active site geometry and thus prevent the conformational 
 change necessary to form the catalytic ternary complex .
we previously 
 observed similar conformational heterogeneity caused by faf bound 
 to 1 nt gap dna in both the absence and presence of pol . the results are also consistent with translesion 
 synthesis studies in which the minor groove conformation benzo[a]pyrene diol epoxide 
n - dg adducts creates steric clash with the active site of pol  , 
 thereby reducing the insertion rate .
these 
 results are in agreement with the steady - state kinetics data that 
 show significant reductions in the fins of dctp opposite fabp in the cg*a and tg*a sequences ( 142- and 59-fold , 
 respectively ) , relative to that of the corresponding unmodified controls .
the question is how to reconcile the apparent lack of discrimination 
 between dntp at the binding step ( figure 7 and 
 tables 3 and 4 ) with 
 the clear preference for accurate insertion of dctp ( figure 4 and tables 1 and 2 ) .
we have recently shown that the af adduct can 
 change its binding characteristics at the replication fork or in a 
 single - nucleotide gap in the active sites of dna polymerases .
similarly , it is plausible that the dynamics 
 of fabp- and faaf - induced conformational heterogeniety could be altered 
 to accommodate an incoming dntp within the active site of polymerases 
 in a way that favors the incorporation of the correct base dctp .
plots 
 of nucleotide specificity ratio ( kd - binary / kd ) with ( a , b ) kf - exo and 
 ( c , d ) pol  for unmodified and modified tg*a and cg*a 
 dna templates .
kd - binary - dg represents kd of 
 unmodified dna - polymerase binary complex , and denominator kd represents the ternary complex of unmodified 
 dna ( or ) binary and ternary complexes of adducted dna . in the present study , we have taken advantage of the 
 sensitivity 
 of spr , which allowed us to probe the delicate interaction between 
 polymerases and dna strands containing arylamine  dna lesions 
 at the binary and ternary complex levels .
we found 
 that 0.73.5 ru of dna coating was sufficient , with no significant 
 interference from mass transport limitation . the binding specificity 
 ratios ( kd of the control binary complex 
 over the kd of a ternary complex ) in the 
 presence of dntps for the unmodified ( dg ) and faaf- and fabp - modified 
 lesions are plotted in figure 7 .
we observed highly specific binding between 
 kf - exo and the native dna substrates in the presence 
 of the correct dctp ( green ) opposite a dg templating base ( figure 7a , b ) .
this is consistent with the polymerase undergoing 
 a conformational change , from open to closed , to form watson 
kf - exo binds weakly with the incorrect 
 dntps , probably retaining the catalytically incompetent open conformation . 

the binding of dgtp ( pink ) with kf - exo was particularly 
 poor .
similar binding results were obtained with pol  ( figure 7c , d ) , although the affinities for modified ternary 
 complexes were generally weaker than those with kf - exo . in both enzymes , however , we observed no discernible nucleotide 
 specificity ( dntps ) and sequence effects ( cg*a vs t*ga ) .
kd values for the ternary complexes 
 for unmodified dna were determined using affinity analysis because 
 the association rate ( ka ) reaches the 
 near - diffusion limit in native dna .
this procedure allowed for the 
 monitoring of interactions between unmodified or adducted dna with 
 different polymerases on a single chip .
the present work also demonstrates 
 the utility of spr in distinguishing the substrate preference of different 
 polymerases ( e.g. , ds / ss vs 1 nt gap for pol  ) . to our knowledge , 
 this is the first comprehensive use of spr to probe nucleotide insertion 
 kinetics during the action of a polymerase .
furthermore , the present 
 spr work advances the limits of spr technology , demonstrating that spr can measure subnanomolar affinity differences 
 between incoming nucleotides and the active site of a polymerase . in conclusion , we have characterized the spr binding affinity of 
 the mutagenic fabp and faaf lesions bound to kf - exo and pol . kf - exo binds strongly to ds / ss 
 template / primer dna , whereas pol  prefers gapped dna .
tighter 
 binding was observed between unmodified dg and kf - exo or pol . the systems exhibited nucleotide selectivity , with kd values increasing in the order of dctp  
 dttp  datp  dgtp . unlike pol  , kf - exo binds tightly to both faaf and fabp lesions in the binary systems . 

the relative insertion efficiency fins of dctp opposite fabp was significantly 
 higher in the tg*a sequence compared to that in the cg*a sequence 
 and the unmodified controls .
while the lesion effect was not significant 
 in kf - exo , the active site of pol  is sensitive 
 to the faaf - induced conformational heterogeneity .
our spr data are 
 complemented by primer steady - state kinetics and f nmr 
 data and provide valuable insights into how lesion - induced conformational 
 heterogeneity in dna alters the action of polymerases and thus affects 
 the nucleotide insertion efficiency and coding potential .
 surface plasmon resonance ( spr ) was 
 used to measure polymerase - binding 
 interactions of the bulky mutagenic dna lesions n-(2-deoxyguanosin-8-yl)-4-fluoro-4-aminobiphenyl 
 ( fabp ) or n-(2-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene 
 ( faaf ) in the context of two unique 5-flanking bases ( cg*a and tg*a ) . 
 the enzymes used 
 were exo - nuclease - deficient klenow fragment ( kf - exo ) or polymerase  ( pol  ) . 
 specific binary and ternary 
 dna binding affinities of the enzymes were characterized at subnanomolar 
 concentrations . 
 the spr results showed that kf - exo binds strongly to a double strand / single strand template / primer 
 junction , whereas pol  binds preferentially to double - stranded 
 dna having a one - nucleotide gap . both enzymes exhibited tight binding 
 to native dna , with high nucleotide selectivity , where the kd values for each base pair increased in the 
 order dctp  dttp  datp  dgtp . in contrast 
 to that for pol  , kf - exo binds tightly to 
 lesion - modified templates ; however , both polymerases exhibited minimal 
 nucleotide selectivity toward adducted dna . 
 primer steady - state kinetics 
 and 19f nmr results support the spr data . 
 the relative 
 insertion efficiency fins of dctp opposite 
 fabp was significantly higher in the tg*a sequence 
 compared to that in cg*a . 
 although kf - exo was not sensitive to the presence of a dna lesion , 
 faaf - induced conformational heterogeneity perturbed the active site 
 of pol  , weakening the enzyme s ability to bind to faaf 
 adducts compared to fabp adducts . 
 the present study demonstrates the 
 effectiveness of spr for elucidating how lesion - induced conformational 
 heterogeneity affects the binding capability of polymerases and ultimately 
 the nucleotide insertion efficiency . 
Introduction
Materials and Methods
Results
Discussion