
				 dna polymerases are organized into seven families : a , b , c , d , x , y , and reverse transcriptase     [ 1 , 2 ] . among these families ,
dna polymerases are involved in dna replication , dna repair , dna lesion bypass , antibody generation , and sister   chromatid cohesion . despite these diverse roles , dna polymerases catalyze the nucleotidyl transfer reaction using a two divalent metal ion mechanism   with at least one positively charged residue   that functions   as a general acid   at their active site , follow a similar minimal kinetic pathway , and share a similar structural architecture consisting of the fingers , palm , and thumb subdomains     [ 8 , 9 ] .
surprisingly , the polymerization fidelity of eukaryotic dna polymerases spans a wide range : one error per one to one billion nucleotide incorporations ( 10 to10 ) .  
the y - family dna polymerases are known for catalyzing nucleotide incorporation with low fidelity and poor processivity .
these enzymes are specialized for translesion dna synthesis which involves nucleotide incorporation opposite and downstream of a damaged dna site .
lesion bypass can be either error - free or error - prone depending on the dna polymerase and dna lesion combination . to accommodate a distorted dna substrate , y - family dna polymerases utilize several features : a solvent - accessible   and conformationally flexible active site , smaller fingers and thumb subdomains , an additional subdomain known as the little finger , the little finger and polymerase core domains move in opposite directions during a catalytic cycle , and a lack of 3  5 exonuclease activity .
unfortunately , these features , which facilitate lesion bypass , may also contribute to the low fidelity of a y - family dna polymerase during replication of a damaged or undamaged dna template .
thus , it is important to understand the mechanism and fidelity of the y - family dna polymerases . 

saccharomyces cerevisiae dna polymerase  ( ypol ) , a y - family dna polymerase , is critical for the error - free bypass of uv - induced dna damage such as a cis - syn thymine - thymine dimer     [ 1519 ] . to date , pol remains the only y - family dna polymerase with a confirmed biological function .
ypol is organized   into a polymerase domain , ubiquitin - binding zinc finger ( ubz ) domain , and proliferating cell nuclear antigen- ( pcna ) interacting peptide ( pip ) motif ( figure 1 ) .
x - ray crystal structures of ypol 's catalytic core have been solved alone      as well as in complex with a cisplatin - dna adduct and an incoming nucleotide . due to a lack of structures for full - length ypol
, it is unclear if the c - terminal residues 514632 interact with dna and contribute to the polymerase function of ypol. using pre - steady - state kinetic techniques , we have measured the base - substitution fidelity of full - length and truncated ypol ( figure 1 ) catalyzing nucleotide incorporation into undamaged dna .
in addition , we have determined the dna binding affinity of both full - length and truncated ypol. our results show that the c - terminus of ypol has a minor effect on the dna binding affinity and the base substitution fidelity of this lesion bypass dna polymerase .
materials were purchased from the following companies : [ -p ] atp , mp biomedicals ( solon , oh ) ; biospin columns , bio - rad laboratories ( herclues , ca ) ; dntps , ge healthcare ( piscataway , nj ) ; oligodeoxyribonucleotides , integrated dna technologies , inc .
( coralville , ia ) ; and optikinase , usb ( cleveland , oh ) .
the primer strand 21-mer or blunt - end 16-mer was 5-radiolabeled with [ -p]atp and optikinase . then ,
the 21-mer was annealed to the appropriate 41  mer template ( table 1 ) and the palindromic blunt - end substrates were annealed as described previously .
the catalytic core of ypol ( 1513 ) containing an n - terminal mgssh6ssglvprgsh tag was purified as described previously .
all experiments were performed in reaction buffer a which contained 40  mm tris - hcl ph 7.5 at 23c , 5  mm mgcl2 , 1  mm dtt , 10  g / ml bsa , and 10% glycerol .
a rapid chemical - quench flow apparatus ( kintek , pa , usa ) was used for fast reactions . for burst assays , a preincubated solution of ypol ( 320  nm ) and
5-[p]-labeled d-1 dna ( 480  nm ) was mixed with dttpmg ( 100  m ) . to measure the dissociation rate of the ypoldna binary complex
, a preincubated solution of ypol ( 50  nm ) and 5-[p]-labeled d-1 dna ( 100  nm ) was mixed with a molar excess of unlabeled d-1 dna ( 2.5  m ) for various time intervals prior to initiating the polymerization reaction with dttpmg ( 150 and 400  m for truncated and full - length ypol , resp . ) for 15  s. for single - turnover kinetic assays , a preincubated solution of ypol ( 150  nm ) and 5-[p]-labeled dna ( 30  nm ) was mixed with an incoming dntpmg ( 0.4800  m ) .
reaction products were analyzed by sequencing gel electrophoresis ( 17% acrylamide , 8  m urea , 1  tbe running buffer ) , visualized using a typhoon trio ( ge healthcare ) , and quantitated with imagequant software ( molecular dynamics ) .   
the equilibrium dissociation constant ( kd ) of the ypoldna binary complex was determined using two techniques .
first , an electrophoretic mobility shift assay ( emsa ) was employed by adding increasing concentrations of ypol ( 10450  nm ) into a fixed concentration of 5-[p]-labeled d-1 dna ( 10  nm ) in buffer a. the solution established equilibrium during a 20-minute incubation period .
then , the binary complex was separated from unbound dna using a 4.5% native polyacrylamide gel and running buffer as previously described except the final concentration of tris was adjusted to 40  mm .
increasing concentrations of ypol ( 2300  nm ) were titrated into a fixed concentration of f-8 dna ( 25  nm ) in buffer a ( devoid of bsa ) .
the f-8 dna substrate ( table 1 ) was excited at a wavelength of 312  nm with emission and excitation slit widths of 5  nm .
the emission spectra were collected at 1 nm intervals from 320 to 500  nm using a fluoromax-4 ( jobin jvon horiba ) .
emission background from the buffer and intrinsic protein fluorescence were subtracted from each spectrum . for the pre - steady - state burst assay ,
the product concentration was graphed as a function of time ( t ) and the data were fit to the burst equation ( 1 ) using the nonlinear regression program , kaleidagraph ( synergy software ) : 
 ( 1)[product]=a[1exp  ( k1t)+k2 t ] . 
							
a represents the fraction of active enzyme , k1 represents the observed burst rate constant , and k2 represents the observed steady - state rate constant .  
data for the emsa were graphed by plotting the concentration of the binary complex as a function of enzyme concentration ( e0 ) and fitting it to a quadratic equation ( 2 ) : 
 ( 2)[edna]=0.5(kddna+e0+d0 )  0.5[(kddna+e0+d0)24e0d0]1/2 . 

d 
 0 is the dna concentration .   for the fluorescence titration experiments , a modified quadratic equation ( 3 )
was applied to a plot of the fluorescence intensity ( f ) measured at 370  nm versus enzyme concentration : 
 ( 3)[f]=fmax  + [ ( fmin  fmax  ) 2d0 ]  {(kddna+e0+d0 )  [(kddna+e0+d0)24e0d0]1/2}.fmax   and fmin   represent the maximum and minimum fluorescence intensity , respectively . for the rate of dna dissociation from the binary complex , a single - exponential equation ( 4 )
was applied to a plot of product concentration versus time : 
 ( 4)[product]=a[exp  ( kofft)]+c . 
							
a represents the reaction amplitude , koff is the observed rate constant of dna dissociation , and c is the concentration of the radiolabeled dna product in the presence of a dna trap for unlimited time .   for the single - turnover kinetic assays , a plot of product concentration versus time was fit to a single - exponential equation ( 5 ) to extract the observed rate constant of nucleotide incorporation ( kobs ) : 
 ( 5)[product]=a[1exp  ( kobst ) ] . 
 
to measure the maximum rate constant of incorporation ( kp ) and the apparent equilibrium dissociation constant ( kd ) of an incoming nucleotide , the extracted kobs values were plotted as a function of nucleotide concentration and fit to a hyperbolic equation ( 6 ) : 
 ( 6)[kobs]=kp[dntp](kd+[dntp ] ) . 
  the free energy change ( g ) for a correct and incorrect nucleotide substrate dissociating from the ednadntp complex
, r is the universal gas constant and t is the reaction temperature in kelvin .
previously , transient state   kinetic techniques have been used to characterize full - length ypol at 30c .
therefore , we first performed a burst assay   ( see section 2 ) to ensure that our purified proteins , truncated and full - length ypol ( figure 1 ) , behaved in a similar manner at 23c . compared to wild - type ypol , the truncated construct contains only the polymerase domain ( figure 1 ) .
a preincubated solution of ypol ( 320  nm ) and 5-[p]-labeled 21/41  mer d-1 dna ( 480  nm ) was mixed with dttpmg ( 100  m ) and quenched with edta at various times .
product concentration was plotted as a function of time and was fit to ( 1 ) , since there were two distinct kinetic phases : a rapid , exponential phase and a slow , linear phase ( data not shown ) .
biphasic kinetics of nucleotide incorporation indicated that the first turnover rate was the rate of nucleotide incorporation occurring at the enzyme 's active site while subsequent turnovers ( i.e. , linear phase ) were likely limited by the dna product release step as demonstrated by full - length ypol at 30c   and other dna polymerases     [ 23 , 29 , 30 ] .  
the equilibrium dissociation constant for the binary complex of ypoldna ( kd ) was measured to determine if the c - terminus of ypol affects dna binding affinity ( scheme 1 ) .
for example , varying concentrations of full - length ypol ( 10450  nm ) were incubated with a fixed concentration of 5-[p]-labeled d-1 dna ( 10  nm ) before separating the binary complex from the unbound dna on a native gel ( figure 2(a ) ) . then
, a quadratic equation ( 2 ) was applied to a plot of the binary complex concentration versus ypol concentration which resolved a kd of 16  1  nm ( figure 2(b ) and table 2 ) . under similar reaction conditions ,
the kd of truncated ypol was estimated to be 34  3  nm , a binding affinity ( 1/kd ) value that is 2-fold weaker than that of full - length ypol ( table 2 ) .   to corroborate these estimated kd values , we measured the true kd for the ypoldna complex using a fluorescence titration assay .
an analog of da , 2-aminopurine , was embedded into the 41  mer template of f-8 dna which is identical to 21/41  mer d-8 dna except that 2-aminopurine flanks the 5 end of the templating dc base ( table 1 ) .
the f-8 dna substrate ( 25  nm ) was excited at 312  nm , and the emission spectrum was collected from 320 to 500  nm .
after serial additions of full - length or truncated ypol in independent titrations , a decrease in the fluorescence intensity of f-8 was observed .
these changes in fluorescence intensity at 370  nm were plotted as a function of the ypol concentration and were fit to ( 3 ) to extract a kd equal to 7  4  nm for full - length ypol ( figure 2(c ) ) and 13  5  nm for truncated ypol ( table 2 ) .
these kd measurements were tighter than those determined using emsa , since the fluorescence titration assay allows ypol to associate and dissociate during data collection .
in contrast , emsa does not maintain a constant equilibrium because dissociated ypol can not reassociate with dna during electrophoresis separation .
nonetheless , there was a confirmed ~2-fold difference in the dna binding affinity between full - length and the catalytic core of ypol which indicates that the c - terminal 119 amino acid residues of ypol slightly enhance the binding of   the enzyme to dna .  
next , we directly measured the rate of dna dissociation from the ypoldna complex ( see section 2 ) .
a preincubated solution of ypol ( 50  nm ) and 5-radiolabeled d-1 dna ( 100  nm ) was combined with a 50-fold molar excess of unlabeled d-1 dna for various time intervals before dttp was added for 15 s to allow ample extension of the labeled d-1 dna that remained in complex with ypol. a plot of product concentration versus the incubation time with the unlabeled dna trap ( data not shown ) was fit to ( 4 ) which yielded dna dissociation rates ( koff ) of 0.008  0.001  s and 0.0041  0.0008  s for truncated and full - length ypol , respectively ( table 2 and scheme 1 ) .
interestingly , the rate of dna dissociation from full - length ypol is 2-fold slower than that from truncated ypol , which indicated that the c - terminus of ypol may slightly contribute to this polymerase 's dna binding affinity .    
based on the measured kd from figure 2(c ) and koff values , the apparent second - order association rate constant ( kon = koff / kd ) of the binary complex ypoldna was calculated to be 0.62 and 0.59  m  s for truncated and full - length ypol , respectively ( table 2 ) .
these similar kon values indicate that the slightly stronger dna binding affinity of full - length ypol is mainly due to a slightly slower rate of dna dissociation ( koff ) .
taken together , the data in table 2 suggest that the c - terminal 119 amino acid residues of ypol slightly hinder the dissociation of dna from the binary complex ypoldna .
this hindrance is through either direct physical interactions between the c - terminus of ypol and dna , modulation of the conformation of the polymerase domain by the c - terminus of ypol , or both . since
a pre - steady - state burst was observed for truncated ypol , we continued to investigate the nucleotide incorporation efficiency ( kp / kd ) by measuring the maximum rate of nucleotide incorporation ( kp ) and the apparent equilibrium dissociation constant ( kd ) of an incoming nucleotide under single - turnover conditions . by performing these experiments with ypol in molar excess over dna , the conversion of d - dnan to d - dnan+1 ( scheme 1 )
a preincubated solution of truncated ypol ( 150  nm ) and 5-[p]-labeled d-7 dna ( 30  nm ) was mixed with varying concentrations of datpmg ( 0.480  m ) and quenched with edta at various times ( see section 2 ) . a plot of product concentration versus time was fit to ( 5 ) to extract the observed rate constant ( kobs ) for datp incorporation ( figure 3(a ) ) .
then , the kobs values were plotted as a function of datp concentration and fit to a hyperbolic equation ( 6 ) which resolved a kp of 6.9  0.4  s and an apparent kd of 17  3  m ( figure 3(b ) ) .
the pre - steady - state kinetic parameters for the remaining 15 possible dntp : dn base pair combinations were determined under single - turnover conditions and were used to calculate the substrate specificity constant ( kp / kd ) ,   discrimination factor ( ( kp / kd)correct/(kp / kd)incorrect ) , and fidelity ( ( kp / kd)incorrect/[(kp / kd)correct + ( kp / kd)incorrect ] ) of truncated ypol ( table 3 ) . overall , the base substitution fidelity of truncated ypol was in the range of 10 to 10 which translates into 1 misincorporation per 100 to 10,000 nucleotide incorporations ( table 3 ) .
depending on the mispair , truncated ypol catalyzed a misincorporation with 30- to 2,700-fold ( 640-fold on average ) lower efficiency than the corresponding correct base pair . to better understand the mechanistic basis of truncated ypol 's fidelity , the equation for polymerase fidelity
can be simplified as follows : 
 ( 8)fidelity=(kp / kd)incorrect[(kp / kd)correct+(kp / kd)incorrect](kp / kd)incorrect(kp / kd)correct=[(kp)incorrect(kp)correct]1[(kd)correct(kd)incorrect]1=(rate  difference)1(binding  affinity  difference)1 . 
							
thus , fidelity is inversely proportional to the rate difference and apparent binding affinity difference between correct and incorrect nucleotide incorporation . in general , the mechanistic basis of ypol 's discrimination was due to a 3- to 68-fold ( 18-fold on average ) weaker apparent binding affinity ( 1/kd ) and 5- to 220-fold ( 50-fold on average ) slower rate constant of incorporation for a mismatched dntp .
although all four correct dntps were bound with similarly high affinity ( table 3 ) , mismatched purine deoxyribonucleotides have 2- to 6-fold lower apparent kd values than mismatched pyrimidine deoxyribonucleotides .
because 5-protruding purines have been found to have stronger stacking interactions with a terminal dna base pair than 5-protruding pyrimidines , the difference in apparent kd values suggests that base - stacking interactions between an incorrect dntp and the terminal primer / template base pair da : dt ( table 1 )   play a role on the binding of dntp by truncated ypol. interestingly , we have previously demonstrated that the preferred nucleotide for template - independent nucleotide incorporation catalyzed by dpo4 , another y - family dna polymerase , is datp mainly due to its strong intrahelical base - stacking ability . to further evaluate the role of base stacking
, we first examined if truncated ypol can catalyze template - independent nucleotide incorporation of datp or dptp ( figure 4 ) onto four palindromic , blunt - end dna substrates ( be1 , be2 , be3 , and be4 in table 1 ) .
the base of dptp , a dntp analog , has four conjugated benzene rings but possesses no hydrogen - bonding abilities .
the dna substrates possess all four possible terminal base pairs and each molecule of them can be bound by a single polymerase molecule .
our radioactive experiments showed that truncated ypol was able to incorporate datp and dptp ( data not shown ) .
then , we individually measured the kinetic parameters for datp and dptp incorporation under single - turnover reaction conditions ( table 4 ) .
interestingly , the apparent kd values of datp were 3- to 5-fold smaller with a purine than those with a pyrimidine on the primer 's 3-base , indicating that base stacking is also important for the binding of datp to the binary complex of ypolblunt - end dna .
this base - stacking effect is more dramatic for dptp incorporation onto blunt - end dna because the apparent kd values of dptp are 10- to 80-fold tighter than datp incorporation onto the same blunt - end dna substrate ( table 4 ) .
thus , the binding free energy difference between datp and dptp is 1.4 to 2.6  kcal / mol .
previously , we have obtained a comparable binding free energy difference of 2.3  kcal / mol for similar blunt - end datp and dptp incorporation at 37c catalyzed by dpo4 .
although neither datp nor dptp forms any hydrogen bonds with a template base when bound by ypolblunt - end dna , the bases of these two nucleotides should have different base - stacking interactions with a terminal base pair of a blunt - end dna substrate considering that a dangling pyrene base ( 1.7  kcal / mol ) has previously been found to possess a higher base - stacking free energy than a dangling adenosine ( 1.0 kcal / mol ) .
however , the base - stacking free energy difference ( 0.7  kcal / mol ) between pyrene and adenosine is smaller than the aforementioned binding free energy difference ( 1.42.6  kcal / mol ) between dptp and datp .
one possible source is favorable van der waals interactions between pyrene and active site residues of truncated ypol. in addition , the base - stacking effect and van der waals interactions may stabilize the ternary complex of ypolblunt - end dnanucleotide and facilitate catalysis , leading to much higher kp values with dptp than those with datp ( table 4 ) . due to the differences in kp and apparent kd ,
the substrate specificity values of dptp are 100- to 1,000-fold higher than those of datp with blunt - end dna ( table 4 ) and 10- to 100-fold higher than mismatched datp with regular dna ( table 3 ) .
the base substitution fidelities of full - length and truncated ypol may differ because the c - terminal , nonenzymatic regions may alter the polymerization fidelity .
for example , the proline - rich domain of human dna polymerase  has been shown to upregulate the polymerase fidelity up to 100-fold . to determine if the c - terminus of ypol influences polymerization fidelity , we measured the pre - steady - state kinetic parameters for dntp incorporation into d-1 dna ( template da ) catalyzed by full - length ypol ( table 5 ) .
the fidelity was calculated to be in the range of ( 1.4 to 2.6 )  10 for full - length ypol ( table 5 ) .
relative to the fidelity of truncated ypol with d-1 ( table 3 ) , full - length ypol has a 3-fold higher fidelity .
therefore , the c - terminus of ypol slightly affects the base substitution fidelity . moreover , truncated ypol discriminated between a correct and incorrect dntp by ~30-fold on average based on the kp difference while the discrimination for full - length ypol was ~170-fold on average for incorporation into d-1 dna ( tables 3 and 5 ) .
the incorporation rate constant for correct dttp was ~4  s for both ypol enzymes , but the misincorporation rate was 3- to 23-fold faster for truncated ypol. this rate enhancement for truncated ypol is partially offset by a greater discrimination at the apparent ground - state binding level so that the fidelity of truncated ypol was only 3-folder lower than that of full - length ypol. our above studies demonstrated that the c - terminus of ypol enhances this enzyme 's dna binding affinity and base substitution fidelity by 2- and 3-fold , respectively .
these results suggest that the nonenzymatic , c - terminal region of ypol ( figure 1 ) has a mild impact on the n - terminal polymerase domain and its activity .
this conclusion is inconsistent with previous studies which have qualitatively demonstrated that mutations or deletions in the ubz domain or pip motif do not affect polymerase activity     [ 3537 ] .
however , these reported qualitative assays are not sufficiently sensitive to detect the small perturbation on polymerase activity as described in this paper .
the presence of the c - terminal 119 residues of ypol may either interact with dna , slightly alter the conformation of the polymerase domain , or both ( see above discussion ) , thereby enhancing its dna binding affinity and polymerase fidelity .
the fidelity of several y - family dna polymerases synthesizing undamaged dna has been determined by employing steady - state     [ 3848 ] , pre - steady - state     [ 28 , 30 , 4953 ] , or m13-based mutation assays     [ 39 , 41 , 42 , 45 , 54 , 55 ] . from these studies , the fidelity ranges from 10 to 10 . under steady - state reaction conditions , the base substitution fidelity of ypol and
human pol has been measured to be in the range from 10 to 10and 10 to 10 , respectively     [ 38 , 40 ] , which is similar to our pre - steady - state kinetic results .
consistently , pol displays the highest substrate specificity for the dctp  :  dg base pair under both steady - state and pre - steady - state reaction conditions ( table 3 and unpublished data , brown and suo )     [ 38 , 40 ] .
this may seem surprising , since pol participates in the efficient bypass of uv - induced dna damage such as a cis - syn thymine - thymine dimer ( i.e. , a datp : dt base pair )     [ 1520 , 56 , 57 ] .
however , pol has also been shown to be efficient at bypassing guanine - specific damage such as 8-oxo-7,8-dihydro - dg     [ 58 , 59 ] , 1,2-cis - diammineplatinum(ii)-d(gpg ) intrastrand cross - links     [ 6063 ] , and various n2-dg lesions     [ 64 , 65 ] .   among the four eukaryotic y - family dna polymerases ( i.e. , pol , dna polymerase  , dna polymerase  ( pol ) , and rev1 ) , rev1 exhibits low fidelity on undamaged dna due to its strong preference for inserting dctp     [ 46 , 52 ] while pol has an unusual preference for dgtp : dt mispairs over datp : dt due to hoogsteen base pair formation     [ 51 , 69 ] .
interestingly , the lowest fidelity base pair for truncated ypol was dgtp : dt ( table 3 ) .
the two hydrogen bonds established in the wobble base pair may enhance the catalytic efficiency of ypol since hydrogen bonding is important for the efficiency and accuracy of ypol .
also noteworthy , the truncated versions of eukaryotic y - family dna polymerases have been used for many biochemical studies in literature . based on our quantitative kinetic analysis of ypol , these results suggested the nonenzymatic regions of y - family dna polymerases do not alter the polymerase activity significantly . as a y - family dna polymerase , ypol displays low fidelity on undamaged dna ( tables 3 and 5 ) .
in contrast , replicative dna polymerases in the a- and b - families have a polymerization fidelity that is 13 orders of magnitude greater than the y - family dna polymerases ( table 6 ) . dna polymerases with higher fidelity
are more proficient at using the ground - state binding affinity to discriminate between a correct and incorrect dntp .
the y - family dna polymerases provide little to no discrimination based on the kd difference while replicative dna polymerases discriminate up to almost three orders of magnitude
. this lack of selection in the ground state by the y - family dna polymerases may be due to the relatively loose and solvent - accessible active site which has minimal contacts with the nascent base pair     [ 11 , 21 , 71 ] .
moreover , nucleotide selection by the y - family dna polymerases in the ground state may be mainly governed by watson - crick base pairing , since the calculated g values ( 0.951.7  kcal / mol ) are similar to the free energy differences between correct and incorrect base pairs ( 0.31.0  kcal / mol at 37c ) at the primer terminus based on dna melting studies ( table 6 ) .
however , with g values 3.0  kcal / mol , the replicative dna polymerases harness the additional 2.0  kcal / mol of energy from other sources such as a tight active site or close contacts with the nascent base pair . one common fidelity checkpoint among dna polymerases
these large differences may correspond to different rate - limiting steps ( e.g. , protein conformational change , or phosphodiester bond formation ) during nucleotide incorporation     [ 9 , 30 , 71 ] .
for ypol , kinetic data suggest that correct and incorrect dntps are limited by a conformational step preceding chemistry , although , additional studies are needed to confirm these results .
this work presents the mechanistic basis of the base substitution fidelity of ypol on undamaged dna , which examined all possible dntp : dn base pair combinations for the first time .
ypol discriminates against incorrect nucleotides at both the ground - state nucleotide binding and incorporation steps .
finally , the 119 residues at the c - terminus have a mild impact on the kinetic mechanism of ypol.
 understanding polymerase fidelity is an important objective towards ascertaining the overall stability of an organism 's genome . 
 saccharomyces cerevisiae dna polymerase  ( ypol ) , a y - family dna polymerase , is known to efficiently bypass dna lesions ( e.g. , pyrimidine dimers ) in vivo . using pre - steady - state kinetic methods , we examined both full - length and a truncated version of ypol which contains only the polymerase domain . in the absence of ypol 's c - terminal residues 514632 , 
 the dna binding affinity was weakened by 2-fold and the base substitution fidelity dropped by 3-fold . 
 thus , the c - terminus of ypol may interact with dna and slightly alter the conformation of the polymerase domain during catalysis . 
 in general , ypol discriminated between a correct and incorrect nucleotide more during the incorporation step ( 50-fold on average ) than the ground - state binding step ( 18-fold on average ) . 
 blunt - end additions of datp or pyrene nucleotide 5-triphosphate revealed the importance of base stacking during the binding of incorrect incoming nucleotides . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions