interstrand cross - links 
 are among the most deleterious types of 
 damage that can be generated in cellular dna .
these 
 lesions are complete blocks to dna transcription and replication because 
 the covalent linkage prevents strand separation .
concurrent 
 modification of both strands in the duplex also presents severe challenges 
 to cellular dna repair systems . accordingly , even small numbers 
 of interstrand dna
we recently reported a new structural type 
 of interstrand cross - link 
 derived from the reaction of a dna abasic ( ap ) site with a guanine 
 residue on the opposing strand of the duplex ( scheme 1 ) .
this cross - link is intriguing 
 because ap sites ( 1 , scheme 2 ) 
 are perhaps the most common type of endogenous damage suffered by 
 cellular dna .
ap sites are generated by spontaneous 
 depurination , nitrosative stress , and enzymatic base excision repair 
 processes . as a result ,
dna from normal mammalian tissue carries steady - state 
 levels of 50  000200  000 ap sites per cell .
ap sites are also generated by exposure of dna to a wide variety 
 of mutagens , toxins , and anticancer drugs .
cross - links derived from this abundant lesion could have significance 
 in biology and medicine . in our previous work ,
dg - ap 
 cross - links were observed at 5-cap/5-ag sequences ( duplex a , figure 1 ) in equilibrium yields of 23% , under physiologically 
 relevant conditions .
the evidence supported a mechanism 
 involving attack of the exocyclic n - amino group of guanine 
 on the ap aldehyde ( scheme 1 ) .
two other canonical nucleobases , adenine and cytosine , also contain 
 exocyclic amino groups that have been shown to react with aldehydes .
however , our previous studies provided no indication regarding whether 
 adenine or cytosine can participate in cross - link formation with an 
 ap site in duplex dna .
no cross - linking was 
 observed at the adenine residue directly opposing the ap site in duplex a ( figure 1 ) .
cross - links involving adenine residues have been observed in dna 
 duplexes containing oxidized abasic sites ; however , it is important to recognize 
 that the structures and reactivities of oxidized abasic sites are 
 distinct from that of the native ap site considered here . dna duplexes 
 used in this study .
ap - containing duplexes were generated 
 from the corresponding du - containing duplexes by treatment with udg . 

locations where cross - links form in substantial yield are indicated 
 with a ( red \ ) . in order to grasp the 
 full potential of ap sites to generate cross - links 
 in duplex dna
, we felt it was necessary to examine whether canonical 
 nucleobases other than guanine can forge cross - links with ap sites 
 in dna .
dna cross - link involving the reaction of adenine 
 residues with ap sites in double - helical dna .
the da - ap cross - links 
 described here formed in remarkably high yields ( 1570% ) under 
 physiologically relevant conditions and we present evidence consistent 
 with a cross - linking mechanism involving attack of the exocyclic n - amino group of adenine on the aldehyde group of the ap site . 

the widespread occurrence of ap sites in cellular dna combined with 
 the high yields of cross - links observed here raise the possibility 
 that this naturally occurring , dna - templated reaction can generate cross - links in the genetic material of living cells .
when considering 
 nucleobase sequences in duplex dna that might support formation of 
 da - ap cross - links , we first recognized that any nucleobase involved 
 in ap - derived cross - link formation likely must be located near the 
 ap site .
this is because cross - linking by a distal nucleobase presumably 
 would generate substantial and energetically unfavorable structural 
 distortion in the duplex . with this in 
 mind , we constructed models of ap - containing dna duplexes to determine 
 sequence locations on the opposing strand that would position the 
 n - amino group of adenine in close proximity to the ap 
 site . in these models , we employed the simplifying assumption that 
 ap - containing duplexes retain b - dna - like character .
this approach successfully predicted cross - linking sequences 
 in the case of dg - ap cross - links .
measurments 
 within these models indicated that the exocyclic n - amino 
 groups of both adenine residues opposing the ap site in a 5-apt/5-aa 
 sequence were located near the ap aldehyde residue ( figure 2 ) .
as noted in the introduction , however , our previous studies showed that adenine residues directly 
 opposing an ap site ( e.g. , duplex a ) did not engage in 
 cross - link formation .
overall , this analysis led us 
 to the prediction that an adenine residue offset one base to the 3-side 
 of the ap site might have potential to engage the ap - aldehyde in cross - link 
 formation ( figure 2 ) . molecular model illustrating 
 the proximity of the abasic site aldehyde 
 and n - amino group of 2-deoxyadenosine in a 5-apt 
 sequence in b - dna .
ap sites were produced at defined 
 locations in dna duplexes by 
 treatment of the corresponding 5-p - labeled , 2-deoxyuridine - containing 
 duplexes with uracil dna glycosylase ( udg ) .
efficient formation of ap sites in the dna was confirmed by piperidine 
 treatment to generate the cleavage product 4 ( scheme 2 ; figure 3 , lanes 3 , 7 , and 
 12 ) .
lanes 15 , duplex b ; lanes 69 , 
 duplex c ; and lanes 1014 , duplex d. lanes 1 and 10 contain the p - labeled , uracil - containing 
 oligodeoxynucleotides .
lanes 2 , 6 , and 11 contain the p - labeled udg - treated ( abasic - site - containing ) duplexes without incubation . 

lanes 3 , 7 , and 12 contain the p - labeled , udg - treated 
 ( abasic - site - containing ) duplexes subjected to piperidine workup ( 1 
 m , 95 c , 25 min ) to cleave the ap site .
lanes 4 , 8 , and 13 contain 
 the cross - linking reactions involving incubation of the abasic - site - containing 
 duplexes for 120 h in hepes buffer ( 50 mm , ph 7 ) containing nacl ( 100 
 mm ) at 37 c .
lanes 5 , 9 , and 14 contain the methoxyamine - capping 
 reactions involving incubation of the abasic - site - containing duplexes 
 in hepes buffer ( 50 mm , ph 7 ) containing nacl ( 100 mm ) and ch3onh2hcl ( 2 mm ) at 37 c .
the p - labeled oligodeoxynucleotides were resolved by electrophoresis 
 on a 20% denaturing polyacrylamide gel , and the radioactivity in each 
 band quantitatively measured by phosphorimager analysis .
the numbers 
 on the left side of the image represent our structural assignment 
 of the bands and referring to the structure numbers in schemes 2 and 3 .
incubation of duplex b containing a central 
 5-gapt/5-aac 
 sequence in hepes buffer ( 50 mm , ph 7 , 100 mm nacl ) at 37 c , 
 followed by denaturing polyacrylamide gel electrophoretic analysis , 
 revealed a 15.1  0.5% yield of a slow - migrating band in the 
 region of the gel where the cross - linked duplex was expected to appear 
 ( figure 3 , lane 4 ) .
we also observed bands corresponding to the intact ap - containing 
 oligonucleotide 1 and small amounts of a product inferred 
 to be the elimination product 3 , based upon its gel mobility .
duplexes c and d , equipped with 3-overhanging 
 ends , generated slow - migrating bands whose gel mobility was retarded 
 relative to the putative cross - link generated by duplex b ( figure 3 , lanes 8 and 13 ) . these results 
 provided evidence that the slow - migrating bands were indeed cross - linked 
 duplexes containing both full - length strands of the parent duplex 
 and ruled out cross - link formation by the more strongly electrophilic 
 3-4-hydroxy-2-pentenal-5-phosphate end group 3 .
inclusion of the aldehyde - trapping agent methoxyamine in the reactions 
 substantially inhibited formation of the slow - migrating bands ( figure 3 , lanes 5 , 9 , and 14 ) , consistent with involvement 
 of the ap - aldehyde residue in cross - link formation ( 2 , schemes 2 and 3 ) .
it is noteworthy that cross - link formation 
 in duplexes b  d occurred in good 
 yields ( 1318% ) at physiological ph and did not require conditions 
 of reductive amination ( e.g. , ph 45 and nacnbh3 ) often used to capture the imine products resulting from the reaction 
 of aldehydes and amines .
a time course experiment showed that easily detectable amounts of 
 the cross - link were formed in duplex b within a few hours 
 ( figure s1 ) .
cross - link formation 
 was a robust reaction that was not highly 
 sensitive to reaction conditions .
for example , in duplex b , similar cross - link yields ( 16  3% ) were observed in hepes 
 buffer ( 50 mm , ph 7 ; 100 mm nacl ) , cacodylate ( 50 mm , ph 7 , 100 mm 
 nacl ) , nah2po4/citric acid ( 16.5/1.8 mm , ph 
 7.0 , 100 mm nacl ) , mops ( 50 mm , ph 7 , 100 mm nacl ) , and bis - tris ( 50 
 mm , ph 7.4 , 100 mm nacl ) and in the presence of the biological thiol 
 glutathione ( 1 mm , in hepes 50 mm , ph 7 , 100 mm nacl ) ( figure s2 )
. the formation of low molecular 
 weight imines and n - arylglycosides , 
 analogous to those shown in scheme 3 , are reversible 
 processes .
this made it interesting to examine 
 the stability of the cross - link generated in duplex b. we found that , once formed , the cross - link in duplex b was stable under a variety of conditions including ph 3 ( sodium 
 acetate , 25 mm , 24 c , 30 min ) , ph 10 ( potassium phthalate , 24 
 c , 25 mm , 30 min ) or mild piperidine workup ( 0.1 m , 60 c , 
 15 min ) ( figure s3 ) .
on the other hand , 
 the cross - link was decomposed by heat ( 90 c , 15 min ) or treatment 
 with the aldehyde - trapping reagent methoxyamine ( 200 mm , 30 min , 60 
 c ; figure s3 ) . to identify the site at which the 
 ap - aldehyde was attached to the opposing strand
, we carried out hydroxyl 
 radical footprinting of a cross - linked duplex containing the 5-apt/5-aa 
 sequence .
the longer duplex e ( figure 1 ) was used to move the dna bands of interest away 
 from a region of the gel where bands were obscured by a 
salt 
 flare  . in this duplex , the strand opposing the ap - containing 
 oligodeoxynucleotide was 5-p - labeled .
the cross - link 
 was generated as described above , isolated from the gel , and subjected 
 to cleavage by a mixture of iron - edta - h2o2 . in this type of experiment
, the site of cross - linking appears as 
 an interruption in the  ladder  of strand cleavage products 
 generated by the iron - edta - h2o2 reagent , because 
 cleavages beyond the cross - link yield large , slow - migrating dna fragments 
 that are connected to the opposing strand . in the present case ,
a clear disruption in the ladder of cleavage 
 products was observed at the 5-adenine residue in the 5-gapt/5-aac sequence ( residue a6 in duplex e , figures 1 and 4 ) .
this provided evidence 
 that cross - linking involves attachment of the ap - aldehyde to the adenine 
 residue that is offset one base to the 3-side of the ap site .
hydroxyl 
 radical footprinting of duplex i to locate 
 the site of cross - link attachment .
duplex i contains 
 the same core sequence as that in duplex b. ( a ) lane 
 1 is a maxam 
gilbert g - specific cleavage ( sequencing ) reaction 
 of the labeled oligodeoxynucleotide strand in duplex i. lane 2 is an a+g specific cleavage ( sequencing ) reaction of the 
 labeled oligodeoxynucleotide strand in duplex i. lane 
 3 is the hydroxyl radical footprinting reaction of the single stranded 
 nonuracil containing oligodeoxynucleotide .
lane 4 is the hydroxyl 
 radical footprinting reaction of the slow - migrating , cross - link band 
 generated by incubation of duplex i hepes buffer ( 50 
 mm , ph 7 ) containing nacl ( 100 mm ) at 37 c .
the p - labeled oligodeoxynucleotides were resolved on a 20% denaturing 
 sequencing gel , and the radioactivity in each band quantitatively 
 measured by phosphorimager analysis .
gilbert g - specific 
 cleavage ( sequencing ) reaction of the labeled oligodeoxynucleotide 
 strand in duplex i. ( c ) a+g specific cleavage ( sequencing ) 
 reaction of the labeled oligodeoxynucleotide strand in duplex i. ( d ) hydroxyl radical footprinting reaction of the slow - migrating , 
 cross - link band .
we next examined the 
 effect of substituting different nucleobases 
 for the key adenine residue in the 5-gapt/5-aac sequence .
substitution of this adenine residue in 
 duplex b with a guanine or thymine residue , respectively , 
 gave duplexes f and g that did not produce 
 significant yields of a slow - migrating cross - link band ( figure 5 ) .
similarly , duplex h containing 2-aminopurine 
 at this position did not efficiently generate the cross - link ( figure 5 ) .
the substitution of 2-aminopurine for the adenine 
 residue amounts to the rather subtle relocation of a single exocyclic 
 amino group from the major groove to the minor groove of the dna duplex 
 ( see structures in figure 1 ) .
thus , the failure 
 of duplex h to generate significant yields of cross - link 
 allowed us to infer that the exocyclic n - amino group of 
 da in the 5-gapt/5-aac sequence 
 was intimately involved in the cross - linking reactions described here . 

substitution of cytosine for adenine at the cross - linking site also 
 did not yield cross - links ( figure s4 ) . 
 cytosine places an exocyclic amino group in the major groove of dna 
 similar to adenine , but the amino group of dc is located 0.7 
  closer to the sugar  phosphate backbone than that of 
 da ( the amino group of da is closer to the helical axis ) .
nucleobase 
 replacement experiments reveal that the underlined adenine 
 residue in the 5-gapt/5-aac 
 sequence of duplex b is critical for cross - link formation . 

replacement of this adenine residue in duplex b with 
 a guanine or thymine residue in duplexes e and f , respectively , abrogates cross - link formation ( lanes 13 
 and 18 ) .
replacement of the crucial adenine residue with 2-aminopurine 
 in duplex g yields only small amounts of a slower - moving 
 band ( lane 8) .
lanes 15 , duplex b , lanes 69 , 
 duplex g , lanes 1014 , duplex e , 
 and lanes 1519 , duplex f. lanes 1 , 10 , and 15 
 contain p - labeled uracil - containing oligodeoxynucleotide 
 duplexes .
lanes 2 , 6 , 11 , and 16 contain the p - labeled 
 udg - treated ( abasic - site - containing ) duplexes without incubation . 

lanes 3 , 7 , 12 , and 17 contain the abasic - site - containing duplexes 
 subjected to piperidine workup ( 1 m , 95 c , 25 min ) to cleave 
 the ap site .
lanes 4 , 8 , 13 , and 18 contain the cross - linking reactions 
 involving incubation of the abasic - site - containing duplexes in hepes 
 buffer ( 50 mm , ph 7 ) containing nacl ( 100 mm ) at 37 c .
lanes 
 5 , 9 , 14 , and 19 contain the methoxyamine - capping reactions involving 
 incubation of the abasic - site - containing duplexes incubated in hepes 
 buffer ( 50 mm , ph 7 ) containing nacl ( 100 mm ) , and ch3onh2hcl ( 2 mm ) at 37 c .
the p - labeled 
 oligodeoxynucleotides were resolved by electrophoresis on a 20% denaturing 
 polyacrylamide gel and the radioactivity in each band quantitatively 
 measured by phosphorimager analysis .
the results described above were broadly consistent 
 with a cross - linking mechanism involving reversible reaction of the 
 exocyclic n - amino group on da with the ap aldehyde to 
 generate hemiaminal , imine , cyclic hydroxyalkylhemiaminal , or enamine 
 linkages at 5-gapt/5-aac sequences 
 in duplex dna ( structures 6 - 9 ) .
maldi - ms 
 analysis of the cross - linked duplex b gave a strong [ m - h ] signal at m / z 12718.66 . 

this value is consistent with the imine 7 , cyclic hydroxylalkylhemiaminal 8 , or enamine 9 ( figure s5 ; calcd [ m  h ] = 12719.4 , 20 ppm dev ) . 

literature precedents describing the properties of structurally related 
 small molecules indicate that the cyclic hydroxylalkylhemiaminal structure 8 likely is favored .
lc - ms / ms analysis provided 
 additional insight regarding the chemical structure of the da - ap cross - link . 

the cross - linked duplex b ( without the 5-p label ) was excised from a 20% denaturing polyacrylamide 
 gel , eluted from the gel slice , digested with a four - enzyme cocktail 
 consisting of nuclease p1 , alkaline phosphatase , and phosphodiesterases 
 i and ii , and subjected to lc - ms / ms analysis using previously reported 
 experimental conditions . selected - ion chromatograms revealed 
 two peaks eluting at 24.1 and 26.1 min displaying the relevant m / z 368252 transition anticipated 
 for the neutral loss of 2-deoxyribose from the putative da - ap cross - link 
 ( figure 6 ) .
further cleavage of the m / z 252 ion gave rise to a dominant fragment 
 ion at m / z 136 in ms / ms / ms ( figure s6 ) , which is identical to the ms / ms observed 
 for the [ m + h ] ion of da , suggesting that the initial m / z 368252 transition arises via 
 loss of the cross - linked 2-deoxyribose adduct from da ( figure 6c ) .
the presence of multiple peaks in the lc - ms / ms 
 ion chromatogram was consistent with the expectation that the anticipated 
 cross - link remnant resulting from the digestion of 8 will 
 exist as an equilibrating mixture of pyranose and furanose isomers 
 ( scheme s1 ) .
lc - esi - ms and 
 ms / ms analysis of the mixture generated by digestion 
 of the cross - linked oligodeoxynucleotide generated by incubation of 
 duplex b in hepes buffer ( 50 mm , ph 7 ) containing nacl 
 ( 100 mm ) at 37 c .
( a ) selected - ion chromatogram monitoring the 
 neutral loss of 2-deoxyribose from the da - ap cross - link .
( b ) ms / ms 
 arising from the fragmentation of the ion at m / z 368 .
( c ) possible structures for the ions observed at m / z 368 , 252 , and 136 in these experiments .
we examined cross - link formation in duplex i that contained a central 5capt/5-aag sequence . 

this experiment was intriguing to us because , in this sequence context , 
 the ap site has the potential to generate cross - links with either 
 the guanine or adenine residue on the opposing strand ( residues colored 
 red in duplex i shown in figure 1 ) .
incubation of duplex i in hepes buffer ( 50 mm , ph 
 7 , 100 mm nacl ) at 37 c produced a remarkable 71.5  3.7% 
 yield of a slow - migrating band in the region of the denaturing gel 
 where the cross - linked duplexes migrate ( figure 7 ) .
similar to the experiments described above in the context of the 
 5-gapt/5-aac sequence , the 
 yield of the slow - moving band in the 5capt/5-aag sequence 
 also was greatly diminished when the aldehyde - capping reagent methoxyamine 
 was added to the reaction mixtures
. formation rate and stability of 
 the cross - link in this sequence was similar to that described above 
 for the 5-gapt/5-aac sequence ( figures s7s9 ) .
these results along with nanospray 
 tof - ms data ( figure s11 ) confirmed that 
 the slow - migrating band generated in the 5capt/5-aag 
 sequence was an interstrand cross - linked duplex involving the ap - aldehyde 
 residue .
footprinting of a cross - linked duplex containing the 5capt/5-aag 
 core sequence indicated that the cross - link attachment was to the 
 underlined adenine residue in the 5capt/5-aag sequence ( figure s10 ) . 

the footprinting results showed no detectable cross - linking to the 
 dg residue in this sequence .
thus , the remarkable cross - link yield 
 in this sequence appears to result from efficient formation of a da - ap 
 cross - link rather than simultaneous formation of dg - ap and da - ap cross - links .
interstrand 
 cross - link formation in duplex i. lane 
 1 , 5-p - labeled uracil - containing precursor of 
 oligodeoxynucleotide duplex i. lane 2 , 5-p - labeled abasic - site - containing oligodeoxynucleotide duplex i without incubation .
lane 3 , 5-p - labeled 
 abasic - site - containing oligodeoxynucleotide duplex i cleaved 
 by treatment with piperidine ( 1 m , 95 c , 25 min ) .
lane 4 , duplex i incubated in hepes buffer ( 50 mm , ph 7 ) containing nacl 
 ( 100 mm ) at 37 c .
lane 5 , duplex i incubated with 
 ch3onh2 ( 2 mm ) in hepes buffer ( 50 mm , ph 7 ) 
 containing nacl ( 100 mm ) at 37 c .
the p - labeled 
 oligodeoxynucleotides were resolved on a polyacrylamide gel , and the 
 radioactivity in each band quantitatively measured by phosphorimager 
 analysis .
lc - ms / ms analysis of the products 
 generated by nuclease p1 digestion of the cross - linked duplex i revealed 
 the presence of a tetranucleotide with the ap - aldehyde conjugated 
 with the underlined adenine from the 5-capt/5-aag sequence ( figure s12 ) , 
 while the corresponding tetranucleotide with the ap - aldehyde being 
 attached to the guanine residue was not detectable ( data not shown ) . 

additionally , lc - ms / ms analysis of the products formed from the digestion 
 with a four - enzyme cocktail showed that 
 the da - ap remnant is present at a level that is 2 orders of 
 magnitude higher than its dg - ap counterpart ( figures 
 s1314 ) .
these results mesh with the footprinting data 
 described above and indicate that the cross - linking occurs predominantly 
 between the ap - aldehyde and the underlined adenine in the 5-capt/5-aag sequence .
the 
 results described here identified a novel cross - link involving 
 reaction of a dna ap site with an adenine residue on the opposing 
 strand of the double helix .
it is now clear that both dg and da can 
 engage in cross - linking with an ap site and two distinct cross - linking 
 sequence motifs , 5-cap/5-ag and 5-apt/5-aa , have been 
 defined as locations for the formation of ap - derived cross - links .
the difference in the favored locations of da and dg residues in 
 these cross - links , with the cross - linking nucleobase displaced to 
 the 3- and 5-side of the ap site , respectively , is 
 a natural consequence of the helical twist of duplex dna coupled with 
 the fact that the n - amino group of adenine is located 
 in the major groove while the n - amino group of guanine 
 resides in the minor groove ( figure s15 ) .
our results were consistent with a mechanism involving attack 
 of 
 the exocyclic n - amino group of adenine on the aldehyde 
 residue of the ap site to generate a hemiaminal intermediate ( 6 , scheme 3 ) .
the mass spectrometric 
 data is consistent with subsequent dehydration of 6 to 
 give the imine ( 7 ) , cyclic hydroxyalkylhemiaminal ( 8) , or enamine ( 9 ) product .
the detailed chemical 
 structure of the da - ap cross - link remains to be rigorously established 
 using 2d - nmr , but chemical precedents involving structurally related 
 small molecules suggest that the cyclic hydroxyalkylhemiaminal ( 8) likely will be the predominant form .
the substantial 
 stability of the da - ap cross - link generated in duplex b is generally consistent with a cyclic hydroxyalkylhemiaminal 8 , in which the reactive imine group of 7 has 
 been masked by intramolecular attack of the hydroxyl group . despite 
 the stability of the cross - link to many of the conditions examined 
 here , the reaction is reversible , and it will be important to determine 
 whether ap - derived cross - links have sufficient stability to block 
 dna processing enzymes such as helicases and polymerases
. it 
 may be important to emphasize that the da - ap cross - links described 
 here formed in good yields ( 1370% ) under physiologically relevant 
 conditions ( ph 7 , 100 mm nacl ) and did not require conditions of reductive 
 amination ( e.g. , ph 45 and nacnbh3 ) often used 
 to stabilize and increase the yield of the imine products resulting 
 from the reaction of aldehydes and amines . in the sequences examined to date ,
the yields of da - ap cross - links 
 were much higher than those observed for the dg - ap cross - links , when 
 both reactions are conducted under the same , physiologically relevant 
 conditions ( ph 7 , in the absence of nacnbh3 ) .
the sequence 5-capt/5-aag 
 ( duplex i ) that , in principle , can form both dg - ap and 
 da - ap cross - links , in fact , generates a remarkably high yield ( 70% ) 
 of the da - ap cross - link .
it seems likely that , in some duplexes such 
 as i , multiple cross - linked species exist in dynamic 
 equilibrium .
the yield of each cross - linked species at equilibrium 
 will be determined by the inherent abilities of each nucleobase to 
 generate imine linkages with the ap aldehyde and by the ability of 
 the dna duplex to position the amino and aldehyde groups in a manner 
 that favors imine formation .
thus , formation of these ap - derived cross - links 
 is conceptually related to template - directed synthesis with dynamic 
 combinatorial imine libraries , in which equilibrium  sorting 
 processes
 involving reversible imine formation among a mixture 
 of various aldehydes and amines ultimately generate higher yields 
 of the imine products that form the most stable ensembles with a templating 
 molecule present in the reaction .
our results 
 established that the equilibrium yields of the da - ap cross - link can 
 be quite high in some sequence contexts , and future work will systematically 
 explore the effects of sequence variation on cross - link yields .
the widespread occurrence of ap sites along with the facile nature of the cross - linking reactions described 
 here make it interesting to consider the possibility that ap - derived 
 cross - links can form in genomic dna . in cells , processes such as repair and strand cleavage ( as shown 
 in scheme 2 ) will compete 
 with the formation of ap - derived cross - links . as a result
, the majority 
 of ap sites in genomic dna almost certainly do not go forward to form 
 interstrand dna  dna cross - links .
nonetheless , because interstrand 
 cross - links are especially noxious lesions , even low yields of endogenous 
 ap - derived cross - links could prove significant in several diverse 
 areas .
first , endogenous dna damage is thought to be important in 
 the etiology of both human aging and sporadic cancers . because cross - links present severe challenges 
 to replication , transcription , and repair , endogenously formed cross - links 
 could be especially significant .
unrepaired cross - links that block 
 transcription and replication may promote cell death and senescence 
 that contribute to the aging of tissues . at the same time , cellular attempts 
 to repair cross - links are error prone and introduce cancer - causing 
 mutations into the genome .
in addition , the realization that ap sites can generate cross - links 
 in duplex dna may help explain why pathways such as nucleotide excision 
 repair and recombination that are typically associated with the repair 
 of more complex lesions are required for eukaryotic cells to survive 
 the introduction of ap sites into their genome .
finally , ap - derived cross - links could be relevant to difficulties 
 inherent in the amplification and sequencing of ancient dna .
ancient dna samples contain a plethora of lesions 
 including oxidative base damage , ap sites , and strand breaks , but it has been suggested that a high density 
 of interstrand cross - links ultimately limit the ability to amplify 
 and sequence the dna in these samples .
the structural nature of the cross - links in ancient dna has not 
 been elucidated , but ap - derived cross - links are good candidates for 
 these blocking lesions that hinder sample processing .
overall , these findings establish ap - derived interstrand 
 cross - links 
 as a family of dna lesions that can form in diverse sequence contexts . 

studies are currently underway to define the structure , occurrence , 
 biochemical properties , and biological consequences of these lesions .
 the loss of a coding nucleobase from 
 the structure of dna is a 
 common event that generates an abasic ( ap ) site ( 1 ) . 
 
 ap sites exist as an equilibrating mixture of a cyclic hemiacetal 
 and a ring - opened aldehyde . 
 aldehydes are electrophilic functional 
 groups that can form covalent adducts with nucleophilic sites in dna . 
 thus , ap sites present a potentially reactive aldehyde as part of 
 the internal structure of dna . here 
 we report evidence that the aldehyde 
 group of ap sites in duplex dna can form a covalent adduct with the 
 n6-amino group of adenine residues on the opposing strand . 
 the resulting interstrand dna  
 dna cross - link occurs at 5-apt/5-aa 
 sequences in remarkably high yields ( 1570% ) under physiologically 
 relevant conditions . 
 this naturally occurring dna - templated reaction 
 has the potential to generate cross - links in the genetic material 
 of living cells . 
Introduction
Results
and Discussion
Conclusions