hydroxyl radicals , generated by cellular 
 oxidative stress and inflammation , damage nucleobases or deoxyribose sugars , or both 
 in dna . at 2-deoxyguanosines , 
 hydroxyl radical - mediated hydrogen abstraction at the deoxyribose 
 c5-position followed by attack at the guanine c8 carbon forms 
 an n7-centered radical , which may be oxidized to diastereomeric 8,5-cyclo-2-deoxyguanosines 
 ( cdg ) . the 8,5-cyclo-2-deoxyadenosines ( cda ) have also 
 been characterized .
these 8,5-cyclopurine-2-deoxynucleosides have been 
 detected at the nucleotide level , in dna , and cells in vitro , in 
 human urine , and in vivo .
they might contribute to neurologic 
 disease in xeroderma pigmentosum complementation group c ( xp - c ) patients .
they are also believed to play roles in cockayne 
 syndrome , breast and ovarian cancer , and familial mediterranean fever .
it has been reported that s - cdg does not block primer elongation by klenow dna polymerases , 
 and datp is preferentially incorporated opposite the lesion in vitro . however , in escherichia coli , s - cdg blocks dna replication and is refractory 
 to repair .
most 
 are s - cdg  a transitions , although s - cdg  t transversions and low levels of deletions 
 of the 5-neighbor dc are also observed . for
both cdg and cda , the diastereomeric ratio at 
 the c5-position depends on experimental conditions and dna 
 conformation .
computational studies predicted that the incorporation of the cda 
 stereoisomers into dna would result in helical distortions at the 
 lesion site .
both the r- and the s - diastereomers of the 8,5-ca ribonucleoside have been crystallized , 
 and both exhibit the anti conformation about the 
 n - glycosidic bond with o4c1n9c8 = 30 or 27 , respectively .
the fused 
 six - member ring c8n9c1o4c4c5 
 adopts the half - chair conformation with the o4 and c4 
 out of plane .
the ribose adopts the o4-exo ( 0 t ) pseudorotation with p = 289 and m = 48. molecular mechanics 
 calculations predicted that the cda diasetereomers maintain the o4-exo pseudorotation when placed opposite dt in dna .
the nmr data and ab initio calculations suggest 
 that incorporation of the s - cda into di- or trinucleotides 
 does not change the o4-exo pseudorotation .
recently , we reported the structure of 
 the s - cdgdc pair in 5-d(gtgcxtgtttgt)-35-d(acaaacacgcac)-3 , 
 containing the dna sequence of p53 codons 272275 
 ( x = s - cdg , scheme 1 ) .
however , the s - cdg deoxyribose 
 shifts to the o4-exo pseudorotation with p = 280. this altered backbone torsion angles  
 from 50 to 67 and  from 120 to 
 149 , as compared with canonical b - dna
. additionally , the torsion 
 angles  and  are changed from 180 to 87 
 and from 120 to 157 , respectively .
the 
 twist and base pair shift helicoidal parameters are perturbed at the 
 cg and xc base pairs .
the purine ring is anti about the n - glycosidic 
 bond , and the fused six - membered ring adopts the half - chair conformation 
 with o4 and c4 out of plane . here
, we report the structures of the duplexes 
 5-d(gtgcxtgtttgt)-35-d(acaaacaygcac)-3 ( x denotes s - cdg ; y denotes either da or dt , 
 scheme 1 ) .
these model the situation following 
 mis - incorporation of dttp opposite s - cdg , leading 
 to s - cdg  a transitions , or following mis - incorporation 
 of datp opposite s - cdg , leading to s - cdg  t transversions in e. coli .
the s - cdgdt mismatch 
 pair adopts a wobble base pairing , providing a plausible rationale 
 for the s - cdg  a transitions . for the s - cdgda mismatch
pair , both s - cdg 
 and da intercalate , but no hydrogen bonding is observed between s - cdg and da .
this is consistent with the lower levels of s - cdg  t transitions in e. coli .
the oligodeoxynucleotide 5-d(gtgcxtgtttgt)-3 ( x = s - cdg ) was 
 synthesized and characterized as reported.the oligodeoxynucleotides 5-d(gtgcgtgtttgt)-3 and 
 5-d(acaaacaygcac)-3 ( y = da or dt ) were synthesized and purified by anion - exchange 
 chromatography ( midland certified reagent co. , midland , tx ) .
the oligodeoxynucleotides 5-d(gtgcgtgtttgt)-3 
 or 5-d(gtgcxtgtttgt)-3 were 
 annealed at 1:1 stoichiometry with the complementary oligodeoxynucleotide 
 5-d(acaaacaygcac)-3 in 10 mm 
 nah2po4 , 100 mm nacl , and 50 m na2edta ( ph 7.0 ) .
the solutions were heated to 95 c for 
 10 min and cooled to room temperature .
the duplexes were isolated 
 using dna grade hydroxylapatite with a gradient from 10 to 200 mm 
 nah2po4 in 100 mm nacl and 50 m na2edta ( ph 7.0 ) and desalted using sephadex g-25 . melting temperatures of the dna 
 duplexes
were measured by uv / vis spectroscopy at 260 nm in 10 mm nah2po4 , 100 mm nacl , and 50 m na2edta ( ph 7.0 ) .
the thermal 
 scans proceeded from 10 to 80 c with an interval of 1 c . 

samples 
 for the nonexchangeable protons were dissolved in 500 l in 
 10 mm nah2po4 , 100 mm nacl , and 50 m 
 na2edta ( ph 7.0 ) .
they were exchanged with d2o and suspended in 280 l of 99.996% d2o , and the 
 ph was adjusted with dilute dcl or naod .
magnitude correlated spectroscopy 
 ( cosy ) spectra were recorded with 512 real data in the t1 dimension 
 and 2048 real data in the t2 dimension .
total correlation spectroscopy 
 ( tocsy ) spectra were recorded with a mixing time of 80 ms .
the exclusive correlation spectroscopy 
 ( e - cosy ) spectra were recorded with 1024 real points in the t1 dimension 
 and 4096 real points in the t2 dimension .
the spectra were zero - filled during processing to create a matrix 
 of 2048  16384 points .
nuclear 
 overhauser effect spectroscopy ( noesy ) spectra were recorded with 
 512 real points in the t1 dimension and 2048 real points in the t2 
 dimension .
noesy spectra were zero - filled during processing to create 
 a matrix of 1024  1024 real points .
noesy experiments used time - proportional 
 phase increment ( tppi ) quadrature detection and mixing times of 60 , 150 , 200 , and 250 ms .
the relaxation delay 
 was 1.5 s. data were processed using the program topspin and analyzed with the program sparky .
samples for the observation of exchangeable 
 protons were dissolved in 500 l of 10 mm nah2po4 and 100 mm nacl , 50 m edta ( ph 7.0 ) containing 9:1 
 h2o : d2o ( v / v ) ( ph 7.0 ) .
the watergate sequence was used 
 for water suppression , with a nuclear 
 overhauser enhancement ( noe ) mixing time of 250 ms .
the p  h experiments were carried out at the h frequency of 600 mhz .
h3 j couplings were applied to determine the phosphodiester 
 backbone conformation.p 
 chemical shifts were referenced using indirect shift ratios .
integration 
 footprints were defined using noe cross - peaks obtained at a mixing 
 time of 250 ms .
noe intensities from data obtained at mixing times 
 of 60 , 150 , 200 , and 250 ms to check for the presence of spin diffusion 
 effects were determined by volume integrations . for each mixing time , 

these were combined as necessary with intensities generated from complete 
 relaxation matrix analysis of a starting structure to yield a hybrid 
 noe intensity matrix .
the program mardigras iteratively refined the hybrid intensity matrix and optimized agreement 
 between calculated and experimental noe intensities .
the randmardi 
 algorithm carried out iterations , randomizing 
 peak volumes within limits specified by the input noise level .
calculations were initiated using isotropic 
 correlation times of 2 , 3 , and 4 ns .
analysis of these data yielded 
 distance restraints used in restrained molecular dynamics ( rmd ) calculations 
 ( table s3 in the supporting information ) and the corresponding standard deviations for the distance restraints . the pseudorotations ( p ) were estimated by examining the jhh of deoxyribose protons .
the data were fit to curves relating the coupling 
 constants to p , deoxyribose pucker amplitude (  ) , and the percentage 
 s type conformation .
the p and  ranges were converted to the 
 dihedral angles 04 . to 
 obtain backbone torsion angle restraints for the modified , flanking , 
 and terminal base pairs , coupling constants measured from h 
p heternuclear multiple bond correlation spectroscopy 
 ( hmbc ) spectra were applied to the karplus relationship to determine the dihedral angle  ( c4c3o3p ) , related 
 to the h3c3o3p angle by a 120 shift .
the  ( c3o3p  o5 ) torsion 
 angles were calculated from the correlation between  and  
 in b - dna . at all other base pairs ,
crick hydrogen - bonding restraints 
 minimized propeller twisting between base pairs , except at the xt base pair in the s - cdgdt mismatched duplex and at the xa and at base pairs in the s - cdgda mismatched duplex .
the partial charges 
 for the cdg nucleotide were obtained from density functional theory 
 ( dft ) calculations , utilizing the b3lyp/6 - 31 g * basis set and the program 
 gaussian .
the starting structures were 
 generated from a and b type dnas by constructing a bond between g5 
 c8 and g c5 followed by 200 iterations of potential 
 energy minimization using the conjugate gradients algorithm .
the generalized 
 born ( gb ) model with parameters developed 
 by tsui and case was used for implicit 
 water simulation .
the program complete relaxation matrix analysis 
 ( corma ) was utilized to calculate the noe intensities from the structures 
 emergent from calculations .
the duplex containing the s - cdgdt base pair exhibited a melting temperature 
 ( tm ) of 38  1 c .
the unmodified 
 dgdt mismatched duplex exhibited a tm of 43 c under the same conditions ( figure 1a ) .
thus , the incorporation of s - cdg reduced 
 the tm by 5 c .
the duplex containing 
 the s - cdgda pair exhibited a tm of 31  1 c .
the unmodified dgda mismatched 
 duplex exhibited a tm of 39  1 c 
 ( figure 1a ) .
h nmr spectra of the duplex containing the s - cdgdt pair were compared with the unmodified mismatched duplex 
 at different temperatures ( figure 1b ) . at the 
 xt base pair , the t n3h 

resonance was not observed at 5 c . for the modified duplex , 
 the t n3h resonance broadened at lower temperature than 
 did the other thymine imino resonances .
h nmr spectra 
 of the duplex containing the s - cdgda base pair 
 at different temperatures are displayed in figure 1c .
the imino resonances for base pairs cg , ga , and 
 ta of the unmodified duplex were not 
 observed .
moreover , for the unmodified duplex containing the ga mismatch , the c and c h5  h6 scalar couplings were not observed , and that 
 of c was weak .
( a ) uv melting profiles of the mismatched duplexes 
 as compared with the corresponding unmodified duplexes : duplex containing 
 dgda base pair (  ) , duplex containing s - cdgda base pair (  ) , duplex containing dgdt base 
 pair (  ) , and duplex containing s - cdgdt 
 base pair (  ) .
( b ) h nmr of the mismatched duplex 
 containing the s - cdgdt base pair at different 
 temperatures .
( c ) h nmr of the mismatched duplex containing 
 the s - cdgda base pair at different temperatures . 
 the broad resonance at 13.1 ppm observed at 515 c 
 was unassignable
the nonexchangeable protons 
 of the s - cdg - modified duplex were assigned based 
 upon the noe sequential connectivity of the base proton h6 or h8 dipolar 
 couplings with h1 deoxyribose protons ( figure 2a , b ) .
for the modified strand , the 
 sequential connectivity was observed from g to c. because the s - cdg nucleotide lacked a proton at 
 the c8 carbon , the sequential connectivity exhibited an interruption 
 at x. the x h1 proton was identified 
 at 6.11 ppm ; it exhibited a weak x h1  
 t h6 noe .
the sequential connectivity resumed from t to t. for the modified strand , all of the deoxyribose 
 h1 protons were observed within a narrow chemical shift window , 
 between 5.8 and 6.3 ppm .
noe ( 250 ms ) connectivities of base h8/h6 
 protons with deoxyribose h1 protons of the s - cdg modified duplexes .
( a ) modified strand for the duplex containing 
 the s - cdgdt base pair .
( b ) complementary strand 
 for the duplex containing the s - cdgdt base 
 pair . ( c ) modified strand for the duplex containing the s - cdgda base pair .
( d ) complementary strand for the duplex containing 
 the s - cdgda base pair .
the assignments of x deoxyribose protons 
 were made by analysis of scalar and dipolar couplings .
figure 3a displays a tile plot derived from a noesy spectrum 
 obtained at 60 ms mixing time .
x h1 exhibited 
 dipolar couplings with h2 and h2 ; weak scalar couplings 
 were also observed .
h3 exhibited dipolar couplings with h2 , 
 h2 , and h4 , whereas the scalar couplings were not 
 observed .
the geminal h2 and h2 protons 
 were not resolved . for the remainder of the duplex ,
the h2 , 
 h2 , h3 , and h4 deoxyribose resonances were 
 unequivocally assigned .
the absolute configurations of the geminal 
 h2 and h2 protons were assigned from their noes to 
 h1 and h3. with the exception of the unresolved resonances 
 for x , g , and t , h2 
 exhibited a weaker noe with h1 than did h2 , whereas 
 it exhibited a stronger noe with h3 than did h2. the 
 resonance assignments of the nonexchangeable dna protons are tabulated 
 in table s1 in the supporting information .
expansions of noesy spectra ( 60 ms ) showing the assignment of s - cdg nonexchangeable protons . ( a ) duplex containing s - cdgdt base pair . ( b )
the resonances of the base imino protons were assigned 
 based on sequential connectivity in noesy spectra , and the assignments 
 were supported by noes to the amino protons of watson 
the noe sequential connectivity was observed from t  g to g and from t  g 
t  t  t to g. at the 5-neighbor 
 base pair , g n1h exhibited noes with cn h1 and n h2 . 
 at the 3-neighbor base pair ,
the t n3h resonance 
 exhibited noes with a h2 and an h1 . with the exception of the terminal base pairs , the 
 remaining noes arising from watson 
it exhibited an noe with xnh , which also had an noe with t n3h ( figure 4b ) .
( a ) duplex containing 
 the s - cdgdt base pair ; noe cross - peaks are 
 assigned as follows : a , a h2  g n1h ; 
 b , cnh2  g n1h ; c , cnh1  
 g n1h ; d , an h1  t n3h ; and e , a h2  
 t n3h . ( b )
assignment of x n1h in the duplex 
 containing the s - cdgdt base pair ; noe cross - peaks 
 are assigned as follows : f , t n3h  xnh ; and g , x n1h  
 xnh .
( c ) duplex containing 
 the s - cdgda base pair ; noe cross - peaks are 
 assigned as follows : h , a h2  g n1h ; 
 i , cnh2  g n1h ; and j , cnh1  
 g n1h . the scalar couplings 
 of the 2-deoxyribose h1 protons with the h2 
 and h2 protons were measured from an e - cosy spectrum ( figure 
 s1 in the supporting information ) .
the jh1-h2 and jh1-h2 values 
 for x were 2.1 and 4.9 hz , respectively .
for t , jh1-h2 and jh1-h2 were 5.3 and 8.8 hz , respectively . 
 with the exception of the terminal nucleotides ,
the jh1-h2 for other nucleotides 
 were 810 hz , and the jh1-h2 were 57 hz . the j coupling 
 constants for the deoxyribose protons are tabulated in table s2 in 
 the supporting information .
the p nmr spectrum of the s - cdg containing 
 duplex was compared with the unmodified mismatched duplex ( figure 
 s2 in the supporting information ) . at the s - cdg nucleotide , the p resonance shifted upfield . 

the other p resonances were clustered within a modest 
 chemical shift range , centered in the spectral region characteristic 
 of b - dna .
chemical shifts of the 
 nonexchangeable protons between the s - cdg - containing 
 duplex andthe unmodified mismatched duplex were compared ( figure 5 ) .
remarkable changes were observed at x and the 5- and 3-neighboring nucleotides of the 
 modified strand .
c h2 shifted downfield by 0.72 
 ppm ; x h2 and h2 shifted upfield by 0.21 
 and 0.51 ppm , respectively ; and t h6 , h1 , and 
 h2 shifted downfield by 0.32 , 0.24 , and 0.23 ppm , respectively . 

the chemical shift perturbations for the complementary strand were 
 small , with the exception of a h8 and t h1 , 
 which shifted upfield by 0.24 and 0.31 ppm , respectively . chemical shift 
 perturbations of the duplex containing the s - cdgdt 
 base pair .
a total of 406 distance restraints , 
 including 263 intranucleotide and 143 internucleotide restraints , 
 were calculated from the intensities of noe cross - peaks ( table s3 
 in the supporting information ) .
a total of 21 noes involving the s - cdg protons were used as restraints .
crick base pairing were used , 
 as were 160 empirical torsion angle restraints that were applied to 
 the nonterminal nucleotides .
these were justified based upon nmr data , 
 which suggested that structural perturbations were localized at and 
 adjacent to the lesion site .
weak wobble base pair restraints were 
 used for the xt base pair , and no torsion 
 angle restraints were used for the cg , xt , and ta base pairs .
r1 = |(a0)i1/6  ( ac)i1/6|/|(a0)i1/6| , where a0 and ac are the 
 intensities of observed ( nonzero ) and calculated noe cross - peaks , 
 respectively .
the rmd calculations for the s - cdg - containing 
 duplex were performed from a and b form starting structures .
ten emergent 
 structures , five each for a- and b - dna starting structures , were obtained 
 and minimized with respect to potential energy .
the 
 accuracies of the emergent structures were evaluated by comparison 
 of theoretical noe intensities for the refined structure calculated 
 by the program corma to the experimental 
 noe intensities , to yield sixth root residuals ( r1 ) .
these , as well as the residuals for intra- or internucleotide noes , 
 were consistently < 0.1 ( table 1 ) .
r1 values for each 
 nucleotide were < 0.15 ( figure s3 in the supporting 
 information ) .
thus , the refined structures provided accurate 
 depictions of the noe data . the xt pair adopted the wobble conformation 
 ( figure 6 ) .
the 
 xt pair exhibited a shift of 0.8 
  , displacing c toward the major groove .
this pair 
 exhibited a greater than normal opening of 16.8. typical b - dna 
 pairing and stacking interactions were maintained for the remaining 
 base pairs ( table s4 in the supporting information ) .
the s - cdg deoxyribose was in the o4-exo ,  west  pseudorotation ( figure 8a ) , with p = 280 and m = 47. the heavy atoms n9 , o3 , and c5 
 were axial about the deoxyribose ring .
pseudorotation , with p = 65 and m = 37. consequently , x h2 was farther 
 from the x purine ring as compared to the h2 protons 
 in b - dna , and c4 h2 was proximate to the x purine 
 ring . with the exception of the terminal nucleotides ,
the six - membered ring c8n9c1o4c4c5 
 adopted the envelope ( half boat ) conformation .
helicoidal analysis 
 of the backbone torsion angles ( figure s4 in the supporting information ) showed that for s - cdg , the  angle shifted from 180 to 78. the 
  angle shifted from 50 to 57. perturbations of 
 the  and  torsion angles from 120 to 147 and from 
 90 to 58 , respectively , were also observed . 

there was also a change for the n - glycosidic torsion angle  
 from 120 to 162. for the complementary t , a perturbation of the  torsion angle from 120 to 
 83 was observed .
expanded views of the refined structure of the s - cdg containing duplexes at the lesion site .
( a ) duplex 
 containing the s - cdgdt base pair , viewed from 
 the minor groove .
( b ) duplex containing the s - cdgdt 
 base pair , viewed from the major groove .
( c ) duplex containing the s - cdgda base pair , viewed from the minor groove .
( d ) 
 duplex containing the s - cdgda base pair , viewed 
 from the major groove .
base pairing and base stacking of the refined structures 
 of the s - cdg containing duplex at the lesion site . 

the nonexchangeable protons 
 were assigned based upon the sequential connectivity of the base proton 
 h6 or h8 dipolar couplings with h1 deoxyribose protons ( figure 2c , d).for the modified strand , 
 the noe connectivity was observed from g to c. the connectivity exhibited an interruption at x due 
 to the lack of a proton at the c8 carbon .
the x h1 
 proton was identified at 6.12 ppm ; it exhibited a weak x h1  t h6 noe , suggesting that the distance 
 between these two protons was greater than in b - dna .
the sequential 
 connectivity resumed from t to t. for the 
 modified strand , the deoxyibose h1 protons were observed within 
 a narrow chemical shift window , between 5.8 and 6.3 ppm .
the resonances of a h2 and a h2 appeared 
 at 7.41 and 7.46 ppm , respectively .
in addition , a h2 exhibited an noe with g n1h ( figure 4c ) , suggesting both a and a were intercalated . as expected , both h2 protons 
 exhibited noes with h1 protons in the minor groove ( figure 9 ) .
notably , a exhibited noes with both 
 t h1 and a h1 of the 5-flanking 
 ta base pair but did not exhibit noes 
 with c h1 or g h1 of the 
 3-flanking cg base pair .
expansion of 
 the noesy spectrum ( 250 ms ) of the duplex containing the s - cdgda base pair showing the intercalation of a and a. noe cross - peaks are assigned as follows : a , 
 t h6  t h1 ; b , t h6  x h1 ; c , a h2  
 a h1 ; d , a h2  t h1 ; e , a h2  a h1 ; 
 f , a h2  x h1 ; g , a h2  a h1 ; and h , a h2 
  a h1. the assignments of x deoxyribose protons 
 were made by analysis of scalar and dipolar couplings .
figure 3b displays a tile plot derived from a noesy spectrum 
 at 60 ms mixing time .
x h1 exhibited dipolar couplings 
 with h2 and h2 ; weak scalar couplings were also observed . 

h3 exhibited dipolar couplings with h2 , h2 , 
 and h4 , whereas the scalar couplings were not observed .
for the remainder of the duplex , the h2 , h2 , h3 , 
 and h4 resonances were assigned unequivocally .
the absolute 
 configurations of the geminal h2 and h2 protons were 
 assigned from their noes to h1 and h3. with the exception 
 of the unresolved resonances for g , t , and 
 c , h2 exhibited a weaker noe with h1 
 than did h2 , whereas it exhibited a stronger noe with h3 
 than did h2. the resonance assignments of the nonexchangeable 
 dna protons are tabulated in table s5 in the supporting 
 information .
the resonances of the base imino protons 
 were assigned based on sequential connectivity in noesy spectra and 
 noes to the amino protons of watson  crick base pairs ( figure 4c).the noe connectivity 
 was observed from t  g to g and from g  t  t  t to g. the resonances of x n1h and t n3h were not assigned although a broad 
 resonance was observed at 13.2 ppm at temperatures below 15 
 c .
the assignment failed due to a lack of noe interactions . 
 at the 5-neighbor base pair ,
g n1h exhibited 
 noes with cn h1 and n h2 . with the exception of the terminal base 
 pairs , the remaining noe cross - peaks arising from watson 
the scalar couplings 
 of the 2-deoxyribose h1 protons with the h2 
 and h2 protons were measured from an e - cosy spectrum ( figure 
 s1 in the supporting information ) .
the jh1-h2 and jh1-h2 values 
 for x were 2.7 and 7.2 hz , respectively .
the jh4-h5 was 6.7 hz , whereas 
 the jh3-h4 was not measurable . with the exception of the terminal nucleotides , 

the jh1-h2 for other nucleotides were 810 hz , and the jh1-h2 were 57 hz . the j coupling constants for the deoxyribose 
 protons are tabulated in table s6 in the supporting 
 information .
h3 
 hmbc spectrum . with the exception of x , each exhibited 
 a heteronuclear coupling with h3 of the 5-neighbor 
 nucleotide
the spectrum of the s - cdg - containing 
 duplex was compared with the unmodified mismatched duplex ( figure 
 s2 in the supporting information ) . at the 
 modified nucleotide ,
the other p resonances were clustered within a modest 
 chemical shift range , in the spectral region characteristic of b - dna .
a total of 399 distance restraints , 
 including 260 intranucleotide and 139 internucleotide restraints were 
 calculated from the intensities of noe cross - peaks ( table s7 in the supporting information ) .
a total of 30 noes involving the s - cdg protons 
 were used as restraints . a total of 43 empirical distance restraints 
 arising from watson 
crick base pairing interactions were used , 
 as were 165 empirical torsion angle restraints that were applied to 
 refine the nonterminal nucleotides .
these were justified based upon 
 nmr data , which suggested that structural perturbations were localized 
 at and adjacent to the lesion site .
the data suggested that no base 
 pairing existed at the xa and ta base pairs , so base pairing restraints 
 were not used for these base pairs .
no torsion angle restraints were 
 used for the cg , xa , and ta base pairs .
ten rmd calculations , five each for a- and 
 b - dna starting structures , were performed .
the 10 emergent structures 
 were minimized with respect to potential energy . all converged as 
 indicated by pairwise rmsd comparisons ( table 1 ) .
the accuracies of the emergent structures were evaluated by comparison 
 of theoretical noe intensities calculated for the refined structure 
 by the program corma to the experimental 
 noe intensities to yield sixth root residuals ( r1 ) .
these , as well as the residuals for intra- or internucleotide noes , 
 were consistently less than 0.1 ( table 1 ) .
r1 values for each 
 nucleotide were less than 0.15 ( figure s3 in the supporting information ) .
thus , the refined structures provided 
 accurate depictions of the noe data . both x and a intercalated into the duplex 
 ( figure 6 ) .
consequently , the helical rise 
 values from ct to xa and from xa to ta were greater than normal , 5.4 and 4.6  , 
 respectively .
figure 7c , d shows the base stacking 
 and base pairing at the lesion site .
significant perturbations in 
 shift were observed from base pairs ta to ga , centered at the xa base pair .
the ct base pair exhibited a greater than normal base pair twist of 54. 
 the remaining base pairs exhibited normal base stacking ( table s8 
 in the supporting information ) .
the s - cdg nucleotide was in the o4-exo ,  west  pseudorotation ( figure 8b ) , with p = 264 and m = 
 47. the heavy atoms n9 , o3 , and c5 were axial 
 about the deoxyribose ring .
consequently , x h2 
 was farther from the x purine ring as compared to the 
 h2 protons in b - dna , while c4 h2 was proximate to 
 the x purine ring . with the exception of the terminal 
 nucleotides ,
the six - membered 
 ring c8n9c1o4c4c5 
 adopted the envelope ( half boat ) conformation .
helicoidal analysis 
 of the backbone torsion angles ( figure s4 in the supporting information ) showed that at the lesion site , the 
  angle shifted from the characteristic 180 to 83. 
 the  angle shifted from 50 to 59. perturbations 
 of the  and  torsion angles from 120 to + 157 and 
 from 90 to 75 , respectively , were also observed . 

there was also a change for the n - glycosidic torsion angle  
 from 120 to 143.
. if not repaired , s - cdg blocks dna replication in e. coli and is genotoxic . in 
 sos - induced e. coli , a mutation frequency of 34% 
 is observed .
most mutations are s - cdg  a 
 transitions , although s - cdg  t transversions 
 and a deletion of the 5-neighbor c are also observed .
accordingly , structures in which s - cdg is placed opposite dt or da , representing intermediates leading 
 to s - cdg  a transitions and s - cdg  t transversions , are of interest .
the dg : dt mismatch often exists as a wobble base pair with both bases 
 in the anti conformation .
one might predict that locking s - cdg into the anti conformation about the n - glycosidic bond would 
 be consistent with the formation of a wobble s - cdg : dt 
 mismatch pair , and this appears to be the case .
the formation of a 
 wobble pair is consistent with the upfield shift of the x n1h resonances , which was also observed for the g n1h 
 of the corresponding unmodified duplex ( figure 1b ) .
however , the t n3h resonance was not observed , indicating 
 enhanced solvent exchange at the s - cdgdt wobble 
 pair .
the structural refinement suggests the potential formation of 
 a three - point hydrogen bond among x n1h , xnh2 , and to and a weak hydrogen bond between xo and t n3h ( figure 7 ) .
however , the observation that the s - cdgdt 
 wobble pair exhibits a tm 5 c lower 
 than the corresponding duplex containing a dgdt mismatch pair 
 ( figure 1a ) suggests that the incorporation 
 of s - cdg reduces the stability of dgdt wobble 
 pairing .
the broadening of the t n3h and t n3h resonances as compared to the other thymine imino resonances 
 ( figure 1b ) suggests that the greatest destabilization 
 occurs at the modified xt and 3-neighboring 
 ta base pairs .
the 
 base pair shifts at the cg and xt base pairs are consistent with this conclusion . 

similarly , for the s - cdg : dc pairing interaction , 
 a 9 c decrease in tm was observed 
 relative to the unmodified duplex .
the 
 thermal destabilization of this duplex is likely associated with the 
 shift of the s - cdg deoxyribose to the o4-exo ( west ) pseudorotation , as opposed to the  south  
 pseudorotation ( c2-endo ) observed in b - dna 
 or the  north  pseudorotation ( c3-endo ) in a - dna . moreover , the complementary t deoxyribose shifts to the c4-exo ( north ) pseudorotation , as evidenced by the jh1-h2 and jh1-h2 of 5.3 and 8.8 hz , respectively 
 ( table s2 in the supporting information ) .
the accommodation of the constrained s - cdg nucleotide 
 necessitates helicoidal perturbation of the phosphodiester backbone 
 torsion angles  ,  ,  , and  in the modified 
 strand .
additionally , smaller perturbations of the t phosphodiester 
 backbone torsion angle  in the complementary strand ( figure 
 s4 in the supporting information ) may factor 
 in the reduced stability of the s - cdgdt vs 
 dgdt mispairing interaction .
both s - cdg and da are inserted into the duplex , but they do not engage 
 in hydrogen bonding . instead , helicoidal perturbations of the modified 
 strand allow both to intercalate , creating a gap at the mismatched 
 region .
the absence of the g n1h  a h2 noe agrees with the gap between a and g caused by the intercalation of s - cdg ( figure 6c , d ) .
this is also consistent with the observation 
 that a h2 exhibits noes with both h1 protons 
 of the 3-flanking ta pair , 
 but not with the h1 protons of the 5-flanking cg pair , suggesting a was close 
 to ta but further from cg ( figure 9 ) .
the observation 
 of the a h2  a h2 noe suggests a remains intercalated .
the 8 c decrease of the tm as compared to the duplex containing a dgda 
 mispair is probably related to alterations of the s - cdg phosphodiester backbone torsion angles  ,  ,  , 
 and  and to perturbations of the base pair shift parameters 
 at the cg and xa base pairs , which are necessitated to accommodate the constrained s - cdg o4-exo ( west ) pseudorotation . 

non - natural , non - hydrogen - bonding base pair that demonstrates excellent 
 polymerase activity , with slower rates of extension .
the s - cdgda mismatch is distinct from the dgda mismatch 
 as dgda mismatch pairs are influenced by sequence and ph .
the 
 failure to observe the imino resonances for base pairs cg , xa , and ta of the unmodified duplex is consistent 
 with this notion and suggests an increased rate of exchange of these 
 protons with solvent , perhaps accompanied by structural disorder . 

the dg(anti)da(syn ) pair has been identified in the crystalline 
 state at ph > 7 , while the protonated dg(syn)da(anti ) 
 pair has been identified at ph 6.6 . in solution , 
 the dg(anti)a(anti ) pair is observed at neutral or basic ph 
 conditions .
another type of dg(anti)da(anti ) pairing is associated with 
 tandem dg : da mismatches .
these differences between the s - cdgda 
 and dgda mismatches are attributed to the fact that the s - cdg lesion is locked into the anti conformation about 
 the n - glycosidic bond and the shift of the deoxyribose to the o4-exo ( west ) pseudorotation .
the da(anti)dg(anti )  face - to - face  
 conformation would predict an noe between g n1h and a h2 , which is not observed ( figure 4c ) . the wobble s - cdgdt pair ( figure 6 )
is 
 consistent with the site - specific mutagenesis studies in sos - induced e. coli , showing a preponderance of s - cdg 
  da transition mutations .
because 
 low levels of s - cd g  dt transversions are observed in e. coli , we surmise that low 
 levels of datp are incorporated opposite s - cdg during 
 trans - lesion synthesis .
it has been suggested that klenow dna polymerases 
 insert datp opposite s - cdg .
further studies of templateprimers containing the s - cdg lesion complexed with error - prone polymerases will 
 be of interest .
the low levels of s - cdg  
 dt transversions might reflect the distortion of the s - cdgda mismatch , in which both s - cdg and da 
 are intercalated but do not hydrogen bond ( figure 6 ) .
have reported 
 that a non - natural , non - hydrogen - bonding base pair exhibiting a similar 
 structure in duplex dna demonstrates excellent polymerase activity , 
 with slower rates of extension .
the structures of s - cdg 
 placed opposite da or dt were determined and compared with the structure 
 when s - cdg placed opposite dc .
the s - cdgdt base pair adopted a 
 hydrogen - bonded wobble conformation , while the s - cdgda base 
 pair differed from dgda mispairs  both s - cdg and da were intercalated , but no hydrogen bonding was observed . 
 in each instance , the s - cdg deoxyribose adopted the 
 o4-exo ( west ) pseudorotation and was accommodated by backbone 
 and base - pairing helicoidal perturbations .
collectively , these perturbations 
 may be important in understanding the mutagenicity and genotoxicity 
 of s - cdg .
 diastereomeric 8,5-cyclopurine 2-deoxynucleosides , 
 containing a covalent bond between the deoxyribose and the purine 
 base , are induced in dna by ionizing radiation . 
 they are suspected 
 to play a role in the etiology of neurodegeneration in xeroderma pigmentosum 
 patients . 
 if not repaired , the s-8,5-cyclo-2-deoxyguanosine 
 lesion ( s - cdg ) induces pol v - dependent mutations 
 at a frequency of 34% in escherichia coli . 
 most are s - cdg  a transitions , suggesting mis - incorporation 
 of dttp opposite the lesion during replication bypass , although low 
 levels of s - cdg  t transversions , arising 
 from mis - incorporation of datp , are also observed . 
 we report the structures 
 of 5-d(gtgcxtgtttgt)-35-d(acaaacaygcac)-3 , where x denotes s - cdg and y denotes either da or 
 dt , corresponding to the situation following mis - insertion of either 
 dttp or datp opposite the s - cdg lesion . 
 the s - cdgdt mismatch pair adopts a wobble base pairing . 
 
 this provides a plausible rationale for the s - cdg 
  a transitions . 
 the s - cdgda mismatch pair differs in 
 conformation from the dgda mismatch pair . for the s - cdgda mismatch pair 
 , both s - cdg and da intercalate , 
 but no hydrogen bonding is observed between s - cdg 
 and da . 
 this is consistent with the lower levels of s - cdg  t transitions in e. coli . 
Introduction
Materials and Methods
Results
Discussion
Conclusions