nucleotide excision repair ( ner ) plays a central role in preserving the genome of prokaryotes and eukaryotes .  
this versatile repair system removes structurally and chemically diverse bulky dna lesions , including those induced by exposure to uv light and environmental chemical carcinogens [ 1 , 2 ] .  
the vital importance of this mechanism is demonstrated by several human ner - deficiency syndromes including xeroderma pigmentosum ( xp ) , cockayne syndrome ( cs ) , and trichothiodystrophy ( ttd ) .  
xp , for example , is characterized by high photosensitivity , hyperpigmentation , premature skin ageing , and proneness to developing skin cancer .  
furthermore , the capacity of the ner pathway is important in cancer chemotherapy : ner diminishes   the efficacy of chemotherapeutic agents such as cisplatin , which act via the formation of bulky dna adducts .
a better understanding of the mechanisms of recognition of dna lesions by the ner system may lead to the design of improved chemotherapeutic drugs that can modulate the repair response .  
recent findings reveal that polymorphisms in human ner repair genes have an impact on the repair of dna lesions and cancer   susceptibility [ 6 , 7 ] , as well as on chemotherapeutic efficacy .   
the eukaryotic ner pathway is a biologically complicated process and consists of two sub - pathways with different substrate specificity :   global genome ner ( gg - ner ) [ 9 , 10 ] and transcription - coupled repair ( tcr ) [ 1114 ] .  
both sub - pathways consist of ordered multistep processes , which differ in the early steps , when the dna lesions are recognized , but converge in the later steps .   in gg - ner ,
the focus of our present interest , the whole genome is scanned for bulky lesions to initiate the repair process .
two independent complexes , one involving the xpc / hr23b / centrin 2 proteins [ 1517 ] and the other involving the ddb1/ddb2 heterodimer [ 1821 ] , have been implicated in the early steps of base - damage recognition during ner .  
by contrast , the tcr sub - pathway is activated by a stalled rna polymerase during transcription .  
once the lesion is detected , the two sub - pathways proceed in an essentially identical manner to excise it :   the multisubunit transcription factor .
tfiih , containing helicases xpb , and xpd , is recruited to the lesion site , followed by xpa , the single - strand dna binding protein rpa , and the two nucleases xpg and xpf - ercc1 .  
once assembled , a 2432 oligonucleotide stretch containing the lesion is excised from the damaged strand .
finally , gap resynthesis by dna polymerases  ,  , and    and ligation by dna ligase i complete the ner process .   
one remarkable characteristic of the ner pathway is its ability to excise an astounding variety of chemically and structurally diverse lesions , and the rates of repair can vary over several orders of magnitude .  
however , the differences in the structural and thermodynamic properties of the lesions that control the diverse ner efficiencies have remained elusive .   it has been suggested that the ner factors do not recognize the lesion itself , but rather the local distortions and destabilizations in the dna that are associated with it [ 2430 ] .   a number of different properties of damaged dna that elicit the ner response have been proposed .  
these   include disruption of watson - crick hydrogen bonding [ 24 , 31 ] , kinks in the damaged dna ,   thermodynamic destabilization [ 24 , 29 , 33 ] , diminished base stacking [ 34 , 35 ] , local conformational flexibility , and flipped - out bases in the unmodified complementary strand [ 3740 ] .   a crystal structure of   yeast rad4/rad23
, the homolog of the human ner recognition factor xpc / hr23b , bound to dna containing a cyclobutane pyrimidine dimer , shows that rad4/rad23 inserts a -hairpin through the dna duplex and expels two mismatched thymines in the undamaged strand out of the duplex to bind with the enzyme   ( pdb i d : 2qsg ) .  
this structure suggests that lesions which thermodynamically destabilize the dna duplex and facilitate the flipping of base pairs and the intrusion of the beta - hairpin are good substrates to the ner machinery :   the more locally destabilized the lesion , the better it is repaired .   
the modulation of ner susceptibility for the same lesion by neighboring base sequence context , is however , a relatively unexplored area .  
if a lesion is better repaired in one sequence context than the other , a lesion - induced mutational hotspot could result .   in order to elucidate the relationship between ner efficiency and base sequence - governed dna distortion and destabilization induced by a bulky dna adduct , we have employed as a model system the major lesion derived from the cancer - causing compound benzo[a]pyrene ( b[a]p ) .  
b[a]p is the most well - studied member in a family of ubiquitous environmental pollutants known as polycyclic aromatic hydrocarbons .
the tumorigenic metabolite of b[a]p   is the diol epoxide r7 , t8-dihydroxy - t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ( b[a]pde ) .  
this intermediate reacts with dna and rna ; the most abundantly stable adduct produced in mammalian cells [ 4446 ] is the 10s ( + ) -trans - anti - b[a]p - n - dg adduct ( [ g * ] ) ( figure 1(a ) ) , the focus of our work .   this adduct , unless removed by dna repair mechanisms , is highly mutagenic [ 48 , 49 ] .
we have investigated the identical 10s ( + ) -trans - anti - b[a]p - n - dg adduct in the six sequence contexts shown in figure 1(b ) , utilizing an array of approaches :   ner in human hela cell extracts , ligation and polyacrylamide gel electrophoresis techniques to assess bending properties of the modified duplexes , and structural studies utilizing high resolution nmr methods as well as unrestrained molecular dynamics ( md ) simulations .  
the position of the b[a]p ring system in the b - dna minor groove , directed 5 along the modified strand , was first determined by nmr in the 5-  c[g*]c - i  sequence in 1992 , but sequence - governed structural details as well as dynamic properties remained to be elucidated .   one important motivation   for our work   was to explore the role of nearby guanine amino groups on the structural properties and ner susceptibilities of these duplexes .  
the key difference in these duplexes is the presence and positioning of guanines flanking the [ g * ] , either immediately adjacent to the lesion or beyond :   the b[a]p rings compete for space   with the bulky amino group of guanine on the minor groove side of b - dna , which we anticipated would differentially impact the structures of the damaged duplexes in a sequence context - dependent manner .   a further motivation was to explore the role of differing sequence contexts beyond the lesion that vary in intrinsic flexibility .  
we hypothesized that subtle but critical structural effects governed by sequence context would manifest themselves by impacting ner efficiencies .   
our results determined that sequence context could cause an up to four - fold difference in relative ner susceptibility , with even distant neighbors influencing ner .  
locally disturbed watson - crick hydrogen bonding and flexible bending are two key sequence - governed structural distortions caused by this lesion that the ner machinery appears to recognize with different efficiencies .   more generally , different lesions in varied sequence contexts will cause different kinds of distortions ; thus , the extent of the local thermodynamic destabilization will also vary ; we hypothesize that it is the extent and type of destabilization that determines the relative ner efficiency .

 the 5-  c[g * ]  , 5-  g[g * ] c  , and 5-  i[g * ]  sequences .  
high resolution nmr solution studies have shown that the bulky aromatic b[a]p residue is positioned in the minor groove on the 5-side of [ g * ]   in the 5-  c[g*]g  and 5-  g[g*]c  duplexes ( figure 2 ) .   however , there are sequence - governed differences in some of the structural features .  
duplex ,   nmr studies revealed that the c  :  g base pair on the 5-side of [ g * ] is severely disturbed .  
in the case of the sequence - isomer 5-  g[g*]c  duplex , this perturbance is not observed .  
on the other hand , analyses of md simulations [ 51 , 52 ] based on the nmr data revealed significant   unwinding near the lesion site combined with an anomalously enlarged roll ( figure 3 ) , not observed in the 5-  c[g*]g  duplex .  
duplex , which is a manifestation of a kink    that is highly flexible .  
this flexible bend is caused on a molecular level by the severe untwisting and enlarged roll determined by md from the nmr data : dna bending is largely caused by increased roll , which is correlated with untwisting [ 5557 ] .  
the underlying structural reasons for the disturbed watson - crick hydrogen bond in the 5- 
, the bulky amino group on g20 ( figure 3 ) , which is partner to the c on the 5 side of [ g * ] , is sterically crowded by the b[a]p ring system since both are on the minor groove side , and hence this c5  :  g20 base pair is episodically denatured ( figure 3(a ) ) ;   for the 5-  g[g*]c  case , the b[a]p rings crowd the g6 amino group , and in this case the crowding is relieved by the severe untwisting accompanied by the increased roll , which produces the flexible bend observed by gel electrophoresis
.    investigations with the 5-  i[g*]c  sequence context substantiated the critical role of the guanine amino group since   
i  ( figure 1(b ) ) lacks this group :   the gel electrophoretic manifestation of a flexible bend was abolished .
the nmr data showed conformational heterogeneity in minor groove conformations , and the md simulations showed episodic denaturation of one of the two hydrogen bonds at the i : c base pair , explaining the heterogeneity .
the repair efficiency relative to 5-  c[g*]c - i  , the standard sequence utilized in many nmr and ner studies [ 53 , 58 ] , is 4.1  0.2 , 1.7  0.2 and 1.3  0.2 for the 5-  c[g*]g  , 5-  g[g*]c  and 5-  i[g*]c  duplexes , respectively ( figure 4 ) .  
duplex , dynamic episodic denaturation of watson  crick base pairing flanking the lesion on the 5-side   correlates with the greatest ner susceptibility while the flexible bend in 5-  g[g*]c  is a less pronounced ner recognition signal , and the disturbance to one hydrogen bond in the 5-  i[g*]c  case provides a still lesser signal [ 52 , 53 ] in this series .  

the 5-  c[g * ] c - i  and 5-  t[g * ] t - ii  sequence contexts . the 5-  c[g*]c - ii  and 5-  t[g*]t - ii 
while a single , well - defined minor groove adduct conformation is observed in 5-  c[g*]c  duplexes , in the 5-  t[g*]t - ii  sequence context , the minor groove - aligned adduct conformation is heterogeneous .   furthermore , polyacrylamide gel electrophoresis studies showed that the adduct induces a rigid bend in the 5-c[g*]c - ii 
sequence context , the lesion induces a highly flexible bend [ 59 , 60 ] .  
also , the 5-  t[g*]t - ii  11-mer duplex has a lower thermal melting point than the 11-mer 5- 
duplex   ( the exact difference depends on sequence length )    as expected from the thermodynamic properties of t  :  a and c  :  g watson - crick base pairs   [ 62 , 63 ] .  
molecular insights on these experimental observations   were provided by md simulations for the 5-  t[g*]t - ii  and 5-  c[g*]c - ii  duplexes .
consistent with the conformational heterogeneity observed in the nmr studies   , it was found that the 5-  t[g*]t - ii  duplex is much more dynamic than the 5-  c[g*]c - ii  duplex : the highly dynamic base pair on the 5-side of the lesion exhibits episodic denaturation of one of the two watson - crick hydrogen bonds , in agreement with the partial rupturing of this base pair observed by the nmr methods ; also , the 5-  t[g*]t - ii  duplex shows somewhat increased and more dynamic roll and untwisting compared to the 5-  c[g*]c - ii 
duplex , consistent with the flexible bend observed only for the 5-  t[g*]t - ii  case ; in addition , the b[a]p ring system exhibits greater mobility and the duplex groove dimensions are more variable .  
the differences are accounted for by a coupled series of properties : the intrinsically weaker stacking of t - g compared to c - g steps allows for greater flexibility in the 5-  t[g*]t - ii 
duplex ;   the weaker t  :  a pair , with only two hydrogen bonds , compared to the c  :  g pair , with three bonds , provides enhanced flexibility ; moreover , the absence of guanine amino groups adjacent to the [ g * ] in the 5-  t[g*]t - ii  case allows for greater mobility for the b[a]p ring system .
overall , the greater flexibility of the 5-  t[g*]t - ii  sequence is attributable to the absence of the guanine amino group .   the rates of incision in the human hela cell assay relative to 5-  c[g*]c - i 
is   2.4  0.2 and 1.6  0.2 for   the 5-  t[g*]t - ii  and the 5-  c[g*]c - ii  duplexes , respectively , corresponding to a 1.5  0.2-fold higher - repair efficiency for the 5-  t[g*]t - ii 
case is consistent with the overall enhanced dynamics manifested in various structural properties , notably watson - crick hydrogen bonding and bending .
the  5-  c[g*]c - i   and  5-  c[g*]c - ii 
sequences ( figure 1(b ) ) differ in the   sequences   beyond the nearest neighbors to [ g * ] .    
since different sequence steps are known to be differentially flexible [ 57 , 65 ] , we hypothesized that the same minor groove lesion [ 50 , 64 ] with different distant neighbors would be differentially repaired .  
polyacrylamide gel electrophoresis and self - ligation circularization experiments revealed that the 5-  c[g*]c - ii 
duplex is more bent and suggested that it has more torsional flexibility than the 5-  c[g*]c - i  duplex .  
the key role is played by the unique -c3-a4-c5- segment in the 5-  c[g*]c - ii  duplex .    
duplex originates from the guanine amino group at the c3  :  g20 pair ( figure 5 ) .  
this amino group acts as a wedge to open the minor groove ; facilitated by the highly deformable local -c3-a4- base step , the amino group allows the b[a]p ring system to better bury its hydrophobic surface within the groove walls .
this produces a yet more enlarged minor groove which is coupled with more local untwisting and more enlarged and flexible roll ,   causing the greater bend   in 5-  c[g*]c - ii    ( figure 5 ) .   
the ner efficiencies are 1.6  0.2 times greater in the 5-  c[g*]c - ii  than in the 5-  c[g*]c - i  sequence context   showing that distant neighbors to [ g * ] modulate the ner susceptibility .   the greater ner susceptibility for the 5-  c[g*]c - ii 
duplex is explained by its greater bending with enhanced flexibility : the intrinsic minor groove enlargement caused by both the guanine amino groups [ 55 , 68 ] and the great flexibility of pyrimidine - purine steps , including the c - a step [ 57 , 6972 ]   allow the b[a]p moiety ( figure 5 )   to more favorably position itself , but at the expense of the greater bend that makes it more repair - susceptible .
we have carried out a series of studies with the same 10s ( + ) -trans - anti - b[a]p - n - dg lesion in a number of sequence contexts that differ in how the lesion is positioned in relation to nearby guanine amino groups .  
additionally , we have considered differences in intrinsic flexibility of sequences flanking the lesion .   these are model systems for gaining understanding of ner lesion recognition factors .  
we have obtained molecular structural data by nmr and md simulations , bending properties from gel electrophoresis studies , and ner data from human hela cell extracts for all of our investigated sequence contexts ( figure 1(b ) ) .  
figure 4 summarizes our key findings and enables us to infer a hierarchy of ner recognition signals for the series of sequences and the single lesion we explored .  
we point out here that a variety of structural disturbances are found in each case , which are correlated .
examples include impaired watson - crick pairing that is accompanied by diminished base stacking , and dna bending towards the major groove , that is induced by a minor groove lesion and is accompanied by minor groove enlargement .  
our present model system suggests that disturbed watson - crick base pairing is a better recognition signal than a flexible bend , and that these can act in concert to provide an enhanced signal : for example , for 5-  t[g*]t - ii  one episodically ruptured watson - crick hydrogen bond combined with the flexible bend results in better repair than   just one disturbed hydrogen bond as in 5-  i[g*]c  , or the flexible bend alone in 5-  g[g*]c    ( figure 4 ) .   for our system , steric hindrance between the minor groove - aligned lesion and nearby guanine amino groups , if present , determines the exact nature of the disturbances , depending on exactly where the guanine amino groups are situated .  
the intrinsic flexibility of the specific base steps also plays an important role in causing the differential disturbances .  
both the nearest neighbor and the more distant neighbor sequence contexts have an impact .   more globally , different lesions may cause different types of distortions depending on the specific nature of the lesion and its sequence context .
however , regardless of exactly what these distortions are , we hypothesize that they must provide a local thermodynamic destabilization signal for repair to ensue , and the greater the extent of destabilization , the better the repair
.   the destabilization would facilitate the strand separation , base - flipping , and -hairpin insertion by the xpc / hr23b recognition factor [ 41 , 73 ] needed to initiate ner .   in this way ,
the ner machinery would excise a large variety of lesions with different efficiencies , by recognizing the thermodynamic impact of the lesions rather than the lesions themselves [ 24 , 29 , 41 , 73 ] .    lesions that resist ner present a great hazard , as they survive to the replication step and produce a mutagenic outcome ; such ner - resistant lesions provide an important opportunity for gaining further understanding of the mechanism utilized by the ner apparatus to recognize different lesions .
 nucleotide excision repair ( ner ) plays a critical role in maintaining the integrity of the genome when damaged by bulky dna lesions , since inefficient repair can cause mutations and human diseases notably cancer .   
 the structural properties of dna lesions that determine their relative susceptibilities to ner are therefore of great interest .   as a model system 
 , we have investigated the major mutagenic lesion derived from the environmental carcinogen benzo[a]pyrene ( b[a]p ) , 
                   10s ( + ) -trans - anti - b[a]p - n2-dg in six different sequence contexts that differ in how the lesion is positioned in relation to nearby guanine amino groups .   
 we have obtained molecular structural data by nmr and md simulations , bending properties from gel electrophoresis studies , and ner data obtained from human hela cell extracts for our six investigated sequence contexts .   
 this model system suggests that disturbed watson - crick base pairing is a better recognition signal than a flexible bend , and that these can act in concert to provide an enhanced signal . 
 steric hinderance between the minor groove - aligned lesion and nearby guanine amino groups determines the exact nature of the disturbances .   both nearest neighbor and more distant neighbor sequence contexts have an impact .   
 regardless of the exact distortions , we hypothesize that they provide a local thermodynamic destabilization signal for repair . 
1. Introduction
2. Nearest Neighbor Base Sequence Context Impacts NER of the  
3.  Distant Neighbor Base  Sequence Context Affects NER of the 
4. Understanding Repairability Differences: the Degree of Local Thermodynamic Destabilization Is a Unifying Hypothesis