more than a decade ago , the unusual catalytic 
 activity of the dna 
 repair enzyme alkb was demonstrated by sedgwick , falnes , and co - workers , whereby 
 oxygen is added to foreign , unnatural carbon atoms attached to nitrogen 
 bases , leading to the liberation of an aldehyde and restoration of 
 the canonical purine or pyrimidine base structures within dna .
initially , 
 the results pointed to a major role for alkb in protecting cells against low molecular weight alkylating agents 
 that react with nitrogen atoms on the watson 
crick base - paring 
 side of purine and pyrimidine dna bases forming covalent adducts , 
 such as the positively charged 1-methyladenine ( m1a ) , 3-methylcytosine 
 ( m3c ) , and 3-ethylcytosine ( e3c ) lesions ( figure 1a ) .
the methylated nucleobases 1-methylguanine ( m1 g ) , 3-methylthymine 
 ( m3 t ) , and n - methyladenine ( m6a ) , uncharged at physiological ph , were also discovered to be substrates 
 of alkb .
more complex dna adducts formed either by oxidative stress - derived 
 lipid byproducts or exogenous vinyl chloride exposure , such as 1,n - ethenoadenine ( ea ) and 3,n - ethenocytosine ( ec ) , as well as 
 the 1,n - ethanoadenine ( ea ) adduct formed 
 by the anticancer agent bcnu ( bis - chloroethylnitrosourea ) , are also 
 repaired by alkb both in vitro and in vivo ( figure 1a ) . although we 
 explored the possibility of alkb acting upon certain alkyl - adducts 
 of oxygen ( o - methylguanine ( omg ) ) and carbon ( 5-methylcytosine ( m5c ) ) , no such evidence 
 was found .
several crystal structures 
 of alkb and homologues bound to alkyl substrates have provided detailed 
 information on their substrate recognition and repair specificity . the substrate scope and properties of alkb
chemical 
 structures and abbreviations of dna lesions investigated 
 as possible repair substrates by the alkb protein using esi - tof mass 
 spectrometry .
( a ) lesions in this panel are known substrates for alkb ; 
 ( b ) lesions in this panel were tested as potential substrates for 
 alkb in this study .
many scenarios of alkylation damage 
 have been explored in order 
 to find possible substrates for alkb with respect to base , position 
 of alkylation , and type of alkyl group damage ( figure 1a ) . however , there are still two positions on the watson  crick 
 base - paring interface of the four bases that , if alkylated , have not 
 yet been investigated as possible substrates for alkb , namely , the n position of guanine and the n atom of cytosine . to provide a complete picture of the 
 substrate range of alkb
, we examined the possibility that this enzyme 
 can act upon the following series of n - alkylguanine adducts : n - methylguanine 
 ( m2 g ) , n - ethylguanine ( e2 g ) , n - furan-2-yl - methylguanine ( ff ) , n - tetrahydrofuran-2-yl - methylguanine ( hf ) , and n - methylcytosine ( m4c ) ( figure 1b ) . the spectrum of lesions chosen for this analysis was motivated 
 by biological relevance gleaned from several studies .
the m2 g lesion 
 is formed by a reaction of the n - amino 
 group of guanine with formaldehyde , a human and rodent carcinogen .
the e2 g lesion is formed by a similar reaction 
 of deoxyguanosine with acetaldehyde , a chemical that occurs both endogenously 
 from the metabolic oxidation of ethanol and from lipid peroxidation 
 products , as well as from environmental sources such as tobacco smoke , 
 vehicle exhaust , and food flavorings .
importantly , acetaldehyde has been shown to be carcinogenic in rodents 
 and can elicit a variety of dna lesions in human cells .
the ff lesion is a structural model of the principal dna adduct 
 formed by nitrofurazone ( nfz ) , a topical 
 antibacterial agent used for treating burns and skin grafts in patients 
 and animals .
postreplicative modification of cytosine 
 by dna methylases produces m4c , which is an epigenetic marker for 
 bacteria . in this study , we used high resolution electrospray ionization time - of - flight 
 ( esi - tof ) mass spectrometry to analyze the reaction products of alkb 
 and the five alkyl adducts mentioned above ( figure 1b ) .
we demonstrate that these lesions are substrates for alkb , 
 leading to the conclusion that all simple n - alkyl 
 lesions , such as methyl adducts , at the watson  crick base - pairing 
 interface on the four bases can be dealkylated by alkb in single stranded 
 dna .
sixteen - mer oligonucleotides 
 of the sequence 5-gaagacctxggcgtcc-3 , 
 where x is the lesion of interest ( m2 g , e2 g , ff , hf , and m4c ) , were 
 synthesized and purified as described .
the calculated monoisotopic molecular weight ( mw ) for each oligonucleotide 
 is shown in table 1 . to create double - stranded substrates , complementary 
 17-mer oligonucleotides 
 of the sequence 5-tggacgccyaggtcttc-3
these were one base longer to avoid overlap with the 
 ms signal from 16-mer lesion - containing oligonucleotides .
y denotes 
 the base placed opposite the lesion x ( c for n - alkylg lesions and g for m4c ) .
dna concentration was measured 
 by uv absorbance using a calculated extinction coefficient (  ) 
 at 260 nm .
the extinction coefficient used for an alkylated base was 
 that of its unmodified counterpart , due to the negligible difference 
 between the values in the context of 16-mer dna .
all assays were performed with alkbn11 protein , 
 a truncated version of alkb in which the first 11 residues were deleted . 

alkbn11 protein was purified as described and shown previously to have activity similar to that of 
 the wild - type protein .
reactions were performed at 37 c in 
 45 mm hepes ( ph 8.0 ) , 0.9 mm -ketoglutarate , 67 m fe(nh4)2(so4)2 , and 1.8 mm ascorbate , 
 followed by dry ice storage until esi - tof ms analysis .
a typical reaction 
 was performed with all the necessary cofactors , 5 m lesion - containing 
 dna , and either 2.5 m alkb protein or protein purification 
 buffer in a 10-l volume for 1 h. where indicated , complementary 
 dna ( 6 m ) was added and annealed at 65 c for 5 min to 
 form a dsdna substrate for the alkb reaction .
oligonucleotide analyses 
 were performed on an agilent 6510 esi - tof mass spectrometer ( palo 
 alto , ca ) .
nitrogen 
 gas was set with drying at 10 l / min , with the heated capillary at 
 325 c and the nebulizer set at 15 psig .
liquid chromatographic 
 separations were performed using a zorbax sb - aq column ( 2.1  
 150 mm ; 3.5 m ; agilent technologies , palo alto , ca ) with a 
 flow rate of 0.2 ml / min .
solvent a was 10 mm ammonium acetate in water 
 ( ph 7 ) ; solvent b was 100% acetonitrile .
a linear gradient 
 was carried out in the following steps : 98 to 70% a over 30 min , 70 
 to 98% a over 5 min , and 98 to 98% a over 10 min .
lc - ms / ms analyses were conducted on an agilent 6510 
 qtof system equipped with an esi source .
operating parameters were as follows : 
 gas temperature , 350 c ; esi capillary voltage , 4000 v ; nebulizer 
 pressure , 50 psi ; drying gas flow , 10 l / min ; fragmentation energy , 
 20 v. the theoretical m / z values , 
 as illustrated in figure s29 ( supporting information ) , were calculated using mongo oligo mass calculator , v2.06 ( http://library.med.utah.edu/masspec/mongo.htm ) .
the ability of alkb to modify the five alkylated bases was tested in vitro using a previously described experimental procedure .
briefly , n - dg and n - dc adducts were incorporated 
 into 16-mer oligonucleotides in a site - specific manner .
the modified 
 oligonucleotides were then incubated with purified alkb for 1 h at 
 37 c , and the reaction products and intermediates ( scheme 1 ) were analyzed by high resolution esi - tof mass 
 spectrometry .
for each lesion , experiments were conducted both in 
 the presence and absence of alkb with all necessary cofactors .
additionally , 
 the repair experiments were also carried out in the presence of a 
 17-mer complementary strand ; the ng lesions 
 were placed opposite a cytosine , and the m4c lesion was placed opposite 
 a guanine .
the 16-mer oligonucleotides demonstrated a good signal 
 in the 4 charge envelope of the mass spectra ( figure 2 ) . to illustrate the method of analysis
, the molecular 
 weight ( mw ) of the 16-mer carrying an m2 g lesion is calculated as 
 4918.87 da ( table 1 ) ; its monoisotopic peak 
 ( all c , n , o , etc . ) in the 
 4 charge state has a calculated m / z of 1228.71 .
experimentally , we observed a peak at an m / z of 1228.62 ( figure 2a and figure s1 ( supporting information ) ) .
esi - tof mass spectra from incubations 
 of n - alkyl 
 dna adducts with the alkb protein .
data represent the intensity vs 
 mass / charge ( m / z ) values for the 
 4 charge envelopes , with the monoisotopic ( all c , n , o , etc . )
peak labeled above each 
 envelope . calculated and observed monoisotopic molecular weights of 
 oligonucleotides and intermediates present in the alkb reactions
( a ) m2 g ; ( b ) m2 g + alkb ; 
 ( c ) e2 g ; ( d ) e2 g + alkb ; ( e ) ff ; ( f ) ff + alkb ; ( g ) hf ; ( h ) hf + alkb ; 
 ( i ) m4c ; and ( j ) m4c + alkb . using the method described above
, we found that in 1 h , 38% 
 of 
 m2 g is converted to guanine in the presence of alkb ( figure 2b , 4 charge m / z 1225.11 ) , leaving 62% of the m2 g unchanged ( m / z 1228.62 ) ( figure 2b ) .
similarly , 
 alkb converted 64% of the e2 g base to undamaged guanine ( m / z 1225.24 , figure 2d ) in 
 1 h. the oligonucleotide containing ff has an m / z of 1245.25 in the absence of alkb ( figure 2e and table 1 ) .
incubation with alkb 
 resulted in the majority of the starting material being converted 
 into two new species ( figure 2f ) , with masses 
 being consistent with undamaged guanine at m / z 1225.24 and ff-2h at 1244.74 ( scheme 1c ) .
the isotopic distribution pattern observed for the assigned 
 ff-2h structure ( figure s9 , supporting information ) was similar to the ff starting material ( figure s7 , supporting information ) , providing confidence 
 that little to no ff starting material was buried underneath the ff-2h 
 signal .
a possible mechanism for ff transformation is that alkb first 
 oxidizes the furfuryl methylene group to generate an ho - ff intermediate 
 ( scheme 1c ) , mirroring the oxidative hydroxylation 
 mechanism alkb uses on other dna alkyl adducts , such as the formation 
 of ho - m6a from m6a .
the ho - ff intermediate 
 can then either directly decompose to undamaged guanine or lose water 
 to form the imine ff-2h . because ff-2h is a schiff s base , 
 it is susceptible to be hydrolyzed to the undamaged guanine and furfurylaldehyde . 
 in the experiment with hf ,
an m / z of 1246.26 was observed for the starting material in the absence 
 of alkb ( figure 2 g ) .
addition of alkb resulted 
 in four peak envelopes in the mass spectrum ( figure 2h ) .
unreacted starting material ( 35% remaining after 60 min 
 incubation ) was clearly identified at an m / z of 1246.26 , while unmodified guanine ( 8% ) was present 
 at 1225.24 ( figure s11 , supporting information ) .
the peak envelope at 1250.26 ( 56% ) matched the calculated mw of 
 the ho - hf ( 1250.22 , scheme 1d and figure s13 , supporting information ) .
for the ho - hf intermediate , 
 the reaction could proceed either to form a dehydration product hf-2h 
 or to decompose to guanine ( scheme 1d ) .
the 
 fourth peak , albeit a small one at 1% , corresponded to the 
 schiff s base type intermediate hf-2h at m / z of 1245.75 ( figure 2h 
 and figure s12 , supporting information ) , 
 which can be hydrolyzed to the undamaged guanine and unconjugated 
 thf - aldehyde ( scheme 1d ) . for the incubation 
 of m4c without alkb ,
an m / z of 1218.70 
 was observed for the 16-mer oligonucleotide containing m4c ( figure 2i ) , which corresponds well with the calculated m / z 1218.71 ( table 1 ) .
there was another small peak envelope at m / z of 1215.45 ( figure s15 , supporting 
 information ) in the spectrum , which corresponds to the 16-mer 
 containing a uracil base at the lesion site ( calculated m / z 1215.45 , table 1 ) .
the 
 uracil base in the oligonucleotide could be generated from the deamination 
 reaction of m4c . in the presence of alkb , about 10% of m4c
is converted 
 to cytosine after 1 h of incubation ( figure 2j and figure s16 , supporting information , 4 charge m / z 1215.20 ) , 
 with approximately 90% of m4c remaining unchanged ( figure 2j ) .
the mw of the oligonucleotide containing cytosine 
 ( 4864.85 ) is 1 da less than that of the oligonucleotide containing 
 uracil ( 4865.83 ) .
the mw difference between the two oligonucleotides 
 at 4 charge state corresponds to an m / z difference of 0.25 unit , which is clearly demonstrated 
 by the observed m / z of 1215.45 for 
 uracil and 1215.20 for cytosine .
these observations strongly support 
 the conclusion that alkb indeed repairs the m4c adduct to unmodified 
 cytosine . to confirm the identity of the oligonucleotides containing 
 lesions , repair intermediates , and products , ms / ms analysis was performed 
 on each of the 5 alkb reactions . in each case , both the unreacted 
 oligonucleotide and the product oligonucleotide were chosen for collision - induced - dissociation 
 ( cid ) .
the results , presented in detail in the supporting information , confirmed the identity of the corresponding 
 oligonucleotide species ( figures s18s29 and tables s2s12 , supporting information ) .
the results of 
 the repair reactions of the five lesions carried 
 out in the presence of the complementary strand are summarized in 
 table s1 ( supporting information ) .
a control 
 experiment to test repair efficiency in both ssdna and dsdna was setup 
 using the known alkb substrate m1a , which was 20% converted 
 to a in 1 h in both contexts .
unlike the reactions in ssdna , the n - aklyl - g and m4c lesions in dsdna were left 
 essentially untouched by alkb .
this study completes 
 our analysis of the sites on dna that alkb 
 can access in order to repair single stranded dna .
the data , taken 
 together , show that the enzyme repairs alkylation damage at all possible 
 nitrogens involved in the normal base - pairing of guanine , cytosine , 
 adenine , and thymine . here , we show that alkb repairs the simple alkyl 
 lesions m2 g , e2 g , and m4c , as well as the bulky lesions ff and hf 
 when present in ssdna , but not in dsdna .
the modified oligonucleotides 
 were incubated with or without purified alkb enzyme in vitro , with or without the complementary strand , and the reaction products 
 were analyzed by esi - tof mass spectrometry and ms / ms .
these assay 
 conditions were not developed to provide kinetic constants for the 
 alkb reactions .
rather , they show qualitatively whether or not a given 
 lesion has the chemical properties needed to be a substrate . if the 
 lesion is chemically competent for repair , it is introduced into cells 
 in subsequent studies to determine if alkb acts upon the lesion in vivo .
the finding 
 that alkb can act on the m4c dna adduct suggests an 
 additional biological function for alkb , besides its normal role as 
 a dna repair enzyme .
the m4c adduct , which is similar to m6a , is normally 
 not considered a deleterious lesion .
the results 
 presented above suggest that alkb might not only act as a repair enzyme 
 to defend against dna alkyl damage but also play a role in controlling 
 gene expression and/or dna replication and segregation .
the 
 present study also highlights the versatility of alkb in dealing 
 with different sizes of alkyl adducts at the n position of guanine .
the enzyme can modify not only the small 
 alkyl lesions m2 g or e2 g but also the bulky ff and hf lesions .
interestingly , 
 the ff and hf lesions , which are structurally similar to each other , 
 displayed very different amounts of alkb - induced repair intermediates 
 ( figure 2 ) .
conversion of ff to undamaged guanine 
 was also much greater than the conversion for hf .
reasons for these 
 observations may be that the ff lesion possesses an aromatic furan 
 ring , which may aid in oxidation of the aliphatic carbon to form ho - ff 
 ( analogous to the oxidation of a chemically reactive benzylic position ) , 
 as well as dehydration of ho - ff to ff-2h .
also , the furfurylaldehyde 
 released enjoys conjugation between the aldehyde carbonyl group and 
 the aromatic furan ring , which is lacking for the ring - saturated thf - aldehyde 
 released from the abundant ho - hf intermediate .
interestingly , a major 
 intermediate , ff-2h ( scheme 1c ) , consistent 
 with being a schiff s base dehydration product , was much more 
 prevalent for the ff alkb incubation than was hf-2h for the analogous 
 hf - derived incubation .
meanwhile , the amount of ho - ff was very small 
 compared to the amount of ho - hf .
one possible explanation is that 
 the dehydration of ho - ff is driven by the formation of the intermediate 
 ff-2h ( scheme 1c ) , a very stable extensively 
 conjugated system throughout the entire guanine and furan rings .
however , 
 hf-2h ( scheme 1d ) lacks such conjugation leading 
 to the accumulation of larger amounts of ho - hf ( figure 2h ) .
alkb is known to repair lesions , such as m1a and 
 m3c , in both ssdna 
 and dsdna , with ssdna being the preferred substrate .
crick 
 base - pairing interactions , thus facilitating their recognition by 
 alkb and its homologues in both ssdna and dsdna . interestingly , while all the lesions tested in this work were repaired 
 by alkb in ssdna , they were not good substrates for repair by alkb 
 when presented in dsdna ( table s1 , supporting 
 information ) .
one possible explanation for these observations 
 relies on the fact that the n - alkyl - dg 
 and m4c lesions display alkyl - groups on the exocyclic amino groups 
 of dg or dc ; these alkyl groups can rotate in the minor or major groove 
 to allow for unimpeded watson 
several crystal structures of alkb 
 with different dna alkyl lesions in ssdna and dsdna have been published .
we modeled the ng lesions 
 based on the structures of alkb with m1a and ea and modeled the m4c 
 lesion based on the m3c structure . among the different conformers 
 generated from the free - rotation of the exocyclic amino groups in 
 of ng and m4c lesions , we chose the conformers 
 that directed the n - attached lesion carbon closest to the fe(ii ) center . 

the analyses showed that the distance between this carbon and the 
 fe(ii ) center was about 5  , which was similar to the distance 
 between the n - attached lesion carbon and fe(ii ) center in the crystal 
 structures of oligonucleotides containing m1a , m1 g , ea , and m3c with 
 alkb .
on the basis of the 
 structural analyses , the five exocyclic lesions tested in this work 
 should be able to adopt conformations that place the alkyl groups 
 in the vicinity of the catalytic fe(ii ) center of alkb , allowing the 
 enzyme to oxidize and dealkylate the modified bases . while alkb 
 may appear to be a panacea for wiping the nucleobase 
 n - alkylation damage slate clean at all watson 
crick base - pairing 
 positions , the relative extents of alkylation removal will depend 
 not only on the position of the alkylated nitrogen atom for each base 
 but also on other factors , which include the alkb binding specificity 
 for a given lesion , the relative conformation of the alkyl group in 
 the active site , and the rate of decomposition of the oxidized intermediates 
 to products .
further kinetic studies are needed to establish the rank 
 order of alkylation repair by alkb and its homologues for the diverse 
 constellation of their alkyl lesion substrates .
 the 
 alkb enzyme is an fe(ii)- and -ketoglutarate - dependent 
 dioxygenase that repairs dna alkyl lesions by a direct reversal of 
 damage mechanism as part of the adaptive response in e. coli . 
 the reported substrate scope of alkb includes simple dna alkyl 
 adducts , such as 1-methyladenine , 3-methylcytosine , 3-ethylcytosine , 
 1-methylguanine , 3-methylthymine , and n6-methyladenine , as well as more complex dna adducts , such as 1,n6-ethenoadenine , 3,n4-ethenocytosine , and 1,n6-ethanoadenine . 
 
 previous studies have revealed , in a piecemeal way , that alkb has 
 an impressive repertoire of substrates . 
 the present study makes two 
 additions to this list , showing that alkyl adducts on the n2 position of guanine and n4 position of cytosine are also substrates for alkb . using 
 high resolution esi - tof mass spectrometry 
 , we show that alkb has the 
 biochemical capability to repair in vitron2-methylguanine , n2-ethylguanine , n2-furan-2-yl - methylguanine , n2-tetrahydrofuran-2-yl - methylguanine , and n4-methylcytosine in ssdna but not in dsdna . 
 when viewed together with previous work 
 , the experimental data herein 
 demonstrate that alkb is able to repair all simple n - alkyl adducts occurring at the watson  
 crick base 
 pairing interface of the four dna bases , confirming alkb as a versatile 
 gatekeeper of genomic integrity under alkylation stress . 
Introduction
Experimental Procedures
Results
Discussion