nuclear and mitochondrial dna is damaged 
 by radiation , by organic 
 and inorganic chemical agents and by the misdirected activity of enzymes . 

exogenous and endogenous processes that contribute to genomic damage 
 include oxidation , alkylation , and deamination of dna .
adducts arising from nucleic acid damage may cause mutations , jeopardize 
 epigenetic patterns , block dna and rna synthesis , inhibit and alter 
 the coding of mrna transcription and translation , and promote strand 
 breaks .
one example , bcnu ( 1,3-bis(2-chloroethyl)-1-nitrosourea ) , 
 has been used to treat lymphoma , multiple myeloma , and several types 
 of brain cancer . among the toxic dna adducts formed by this chemotherapeutic 
 agent
is 1,n - ethanoadenine ( ea , figure 1a ) , which blocks dna replication . in the ea dna 
 adduct , the exocyclic n - nitrogen of adenine 
 is connected to the n1 ring nitrogen by a saturated two - carbon bridge , 
 creating a five - membered ring involving the n1 and n atoms that otherwise would be involved in canonical 
 watson  crick h - bonding ( figure 1a ) . 

ea is repaired to some extent by the escherichia coli repair protein 3-methyladenine dna glycosylase ( alka ) and the human alkyladenine dna glycosylase mpg ( also called 
 aag , anpg , or apng ) . however , for both 
 of the enzymes , the excision of ea is far less efficient than that 
 of the structurally related 1,n - ethenoadenine 
 ( ea ) , in which the two - carbon bridge is unsaturated ( figure 1d ) . because ea lacks 
 the structural features needed to form a watson 
crick base 
 pair with thymine , it is likely to be both toxic and mutagenic . in 
 the absence of repair ,
ea blocks polymerase bypass and miscodes during 
 attempted replication by mammalian dna polymerases in vitro .
the analogous ea adduct , which is also missing 
 the identical base - pairing capabilities , is both toxic and mutagenic 
 in e. coli in the absence of protection 
 by the alkb repair enzyme .
previously , 
 we reported that alkb can effectively alleviate the toxicity of the 
 ea lesion in vivo ; in alkb - proficient cells , ea is easily tolerated 
 and not significantly mutagenic .
however , 
 ea is extremely toxic to alkb - deficient cells , showing an 86% reduction 
 in replication .
preliminary in vitro experiments 
 done in parallel show that alkb could only partially convert ea to 
 generate an open - ring intermediate 2 ( figure 1a , box ) , trappable with pfbha . in an attempt to 
 reconcile the potent ability of the protein to suppress toxicity in 
 vivo with only partial conversion of the lesion in vitro , we speculated 
 that intermediate 2 , via flexible bond rotation , can 
 take on a structure to form a regular watson  crick base pair 
 with thymine ( figure 1a , box ) . that restructured 
 base pair would not block dna replication and hence enables tolerance 
 of the lesion .
chemical structures of ea , m6a , m1a , and ea and proposed 
 mechanisms 
 of alkb - mediated enzymatic reactions on alkyl - dna adducts . ( a ) ea , 
 ( b ) m6a , ( c ) m1a , and ( d ) ea .
the alkb protein is an -ketoglutarate- and 
 fe(ii)-dependent 
 dioxygenase that can efficiently repair various alkyl lesions in both 
 dna and rna , in single- and double - stranded contexts .
it has at least nine human homologues : abh1 through abh8 and fto , 
 some of which have also been proposed or proven to act upon alkylated 
 dna and rna .
alkb , together 
 with ada , alka , and aidb , is one of the four proteins in the e. coli adaptive response to alkylating agents .
the reported substrate scope for the alkb enzyme 
 includes 1-methyladenine ( m1a ) , 3-methylcytosine ( m3c ) , 3-ethylcytosine 
 ( e3c ) , 1-methylguanine ( m1 g ) , 3-methylthymine ( m3 t ) , 3,n - ethenocytosine ( ec ) , ea , and ea . in the current work ,
we used high - resolution esi - tof 
 mass spectrometry to investigate the 
 ability of alkb to repair ea . to our surprise
we found that the oxidizing 
 enzyme alkb is capable of complete repair of the ea base ( figure 1a ) .
the observation of alkb s complete conversion 
 of ea to adenine along a defined reaction path makes plausible the 
 strategy of designing inhibitors to block the repair activity , thus 
 potentially increasing the anticancer efficacy of bcnu .
the 
 ea dealkylation process requires two oxidation reactions , one 
 at n1 and a second at n , to achieve complete 
 restoration of the undamaged adenine base .
the observation of n oxidation in the reaction of alkb with ea 
 suggested that the enzyme might act on other n - modifications of adenine .
the m6a base is functionally similar to n- and 5-methylcytosine ( m4c and m5c ) , 
 and is an important epigenetic signal for dna replication and repair , 
 protein  dna interactions , host - pathogen interactions and other 
 cellular processes ; there is approximately one m6a for every 200 bases in the e. coli genome .
as controls 
 against which to compare the chemistry of alkb on m6a ( and ea ) , we 
 used m1a and ea ( figure 1c and 1d ) , which are good substrates for alkb both in vivo and in 
 vitro . as we were preparing this manuscript , jia 
 et al . reported
that the obesity - associated fto protein ( one of alkb s 
 mammalian homologues ) can remove the methyl group from m6a in both 
 rna and dna ; they indicated that the demethylation of fto on m6a in 
 mrna correlates with epigenetic regulation in mammalian cells . in our present study
the implications of this discovery include the 
 possibility that alkb might be not only a dna repair enzyme in e. coli but also a participant in cellular epigenetic 
 control , which may help cells defend against alkylation damage to 
 nucleic acids .
this study utilized chemical synthesis to incorporate 
 structurally 
 defined lesions into specific sites of oligonucleotides .
the modified 
 oligonucleotides were allowed to react with purified alkb and snapshots 
 were taken at various time points to identify the products and intermediates 
 of reactions .
this method enabled us to efficiently monitor the ability 
 of alkb to repair alkylation damage using a high - resolution mass spectrometry 
 tool employed by us earlier with a number of dna lesions .
four 16mer oligonucleotides 
 were chemically synthesized containing 
 the alkyl - dna adducts shown in figure 1 .
the 
 sequence used was 5-gaagacctxggcgtcc-3 ; the flanking 
 sequence of each lesion ( x ) was identical , fixing the local sequence 
 context for the repair reaction .
the 16mers with individual lesions 
 were incubated with the cofactors for the alkb reaction : fe(ii ) and 
 -ketoglutarate ( see experimental section ) . in the presence of all cofactors ,
two sets of experiments were 
 conducted for each lesion , one with the alkb protein and one with 
 the purification buffer of alkb but without the protein .
following 
 the repair reaction , hplc - esi - tof mass spectrometry was utilized to 
 identify the products and intermediates .
the 16mer oligonucleotides 
 demonstrated a good signal in the 4 charge envelope of the 
 mass spectra .
to give one example to illustrate the method of analysis , 
 the molecular weight ( mw ) of m1a in the 16mer is calculated as 4902.88 
 da for the neutral species , and the mw of its monoisotopic peak ( all c , n , etc . ) when ionized with four negative 
 charges ( the 4 charge envelope ) in the electric field of the 
 spectrometer is calculated as having an m / z of 1224.71 ( all mws are shown in table 1 ) .
the multiple peaks in each 4 charge envelope 
 reflect the number of c or other heavier isotopes ( figure 2a ) .
keeping with m1a as the example , its monoisotopic 
 peak is observed as 1224.67 , consistent with 
 the theoretical calculation .
the next peak in that envelope has an m / z of 1224.92 , 0.25 amu larger than the 
 1224.67 peak , which indicates a species containing c , n , or another isotope that adds a nominal mass of 1.0 to 
 the total weight of the 16mer ( e.g. , 0.25 = m / z = one c / charge state of 4 ) .
additional peaks 
 in the spectrum represent additional heavy isotopes within the parental 
 16mer .
data represent the 4 charge envelopes and the monoisotopic 
 peak ( all c , n , etc .
( a ) m1a , ( b ) m1a + alkb , ( c ) ea , and ( d ) ea + 
 alkb .
previously , we 
 investigated m1a and ea lesions as substrates for alkb both in vitro 
 and in vivo ; we used those two adducts as controls for the present study on the 
 structurally similar compounds , m6a and ea . in our in vitro 
 study of m1a repair
, the lesion was completely repaired to the undamaged 
 base adenine ( figure 2b , m / z = 1221.13 ) , while no change occurred in the absence 
 of alkb ( figure 2a , m / z 1224.67 ) .
the in vitro results confirm that m1a is a good 
 substrate for alkb , and reproduce our prior findings .
moreover , the current observations correlate very well 
 with the strong reparability of m1a suggested by earlier in vivo lesion 
 bypass studies .
ea was observed at m / z of 1227.22 for its 4 charge monoisotopic 
 peak in the absence of alkb ( figure 2c ) . in 
 the presence of alkb
, the ea lesion was mostly converted to the undamaged 
 product , adenine ( m / z = 1221.24 , 
 figure 2d ) .
we also observed peak clusters 
 consistent in mw with epoxide ( 1231.22 ) and glycol ( 1235.72 ) intermediates 
 ( figure 1d and figure 2d ) , which are consistent with previous observations . in our in vitro experiment 
 with ea , the oligonucleotide with ea in the absence of alkb
was observed 
 at m / z of 1227.67 for its 4 
 charge monoisotopic peak , which agrees well with the calculated m / z 1227.71 ( figure 3a and table 1 ) . in the presence of alkb ,
indeed , ea was converted to four new species in the mass spectrum 
 ( figure 3b ) .
the peak at m / z of 1221.16 corresponds to the 16mer with an undamaged 
 adenine base , which we did not find in our previous study .
one possible reason could be that the maldi mass 
 spectrometry tool we used earlier has poorer resolution than the current 
 hplc - esi - tof method .
another reason is that maldi analyzes the samples 
 immediately after the reactions , giving less time for conversion of 
 intermediates to adenine .
however , the hplc - esi - tof method detects 
 mass signals after the hplc separation at room temperature , which 
 might provide product and intermediate profiles different than those 
 detected in the maldi analyses .
we also controlled the hplc autosampler 
 temperature at 5 c and found the formation of undamaged adenine 
 more limited under this condition ( figure 4 ) .
detailed analyses of the product and intermediate kinetics will 
 be presented in a later section .
we also found peaks at m / z of 1231.66 and 1235.67 , which match up with intermediates 
 with one and two extra oxygen atoms added to the ea motif , respectively . 
 by carefully comparing the high - resolution mws between the new species 
 with m / z of 1228.16 and the 16mer 
 with adenine in the lesion position , we determined that this new species 
 has two extra atoms , consistent with the addition of one oxygen and 
 one carbon ( intermediate 6 or 9 in figure 1a ) .
esi - tof mass spectra from reactions of alkyl adducts and 
 alkb protein . 

data represent the 4 charge envelopes and the monoisotopic 
 peak ( all c , n , etc .
( a ) ea , ( b ) ea + alkb , ( c ) m6a , and ( d ) m6a + 
 alkb . in our previously proposed mechanism ( figure 1a , boxed ) , alkb can oxidize the carbon atom attached 
 to the n1 position 
 of the adenine base .
the mono - oxidized intermediates 1 and 2 ( figure 1a ) equilibrate 
 between the ring - closed and ring - opened forms , both bearing an observed m / z of 1231.66 .
the opened - ring intermediate 2 can take on a conformation in which it can form a watson  crick 
 base pair and thereby not block dna replication .
this hypothesis is 
 strongly supported by in vivo experiments showing ea is neither toxic 
 nor mutagenic in alkb - proficient e. coli cells . in the current study
, we observed 
 ea to be completely repaired to the adenine base , which requires oxidation 
 reactions at both the n1 and n - attached 
 carbon atoms ( figures 1a and 3b ) . a clue as to the reaction path of ea was the observation 
 of an alkb - induced glycol ( m / z of 
 1235.67 ) identical to the glycol formed during removal of the etheno 
 bridge from ea ( figure 1d ) . in order for ea 
 to form a glycol ( intermediate 4 , figure 1a ) such as that of ea
the presence of the 
 glycol and the observation of other intermediates indicated by their 
 masses in figure 1a allowed us to assemble 
 a reaction network that would explain the removal of the ethano - bridge 
 from ea .
as shown in figure 1a , alkb can oxidize 
 each carbon of the saturated two carbon bridge and completely restore 
 the undamaged adenine base .
the first oxidation can occur either at 
 the n1- or n - attached carbon of the ea 
 moiety and generate intermediate 1 or 3 , 
 respectively
. each ring - closed intermediate will equilibrate with 
 its ring - opened form 2 or 7 .
further oxidation 
 of 1 or 3 will generate a common intermediate 4 , which can also exist in the respective ring - opened form 
 of 5 or 8 .
further oxidation of the ring - opened 
 intermediate 2 or 7 will also generate 5 or 8 .
the two - carbon unit attached to ea in 
 intermediate 5 or 8 can then be released 
 as glyoxal to form the undamaged adenine ( figure 1a ) , identical to the final step of ea repair after epoxide 
 hydrolysis ( figure 1d ) .
if the glycol 4 or the -hydroxy aldehyde 
 intermediate 5 or 8 is further oxidized , 
 two aldehyde products will form : the terminal carbon will be released 
 as formaldehyde , and the other carbon attached to either the n- or n1-position of the adenine base will generate 
 intermediates 6 or 9 .
the formation of intermediate 6 or 9 should adopt a mechanism similar to typical 
 oxidative cleavage of a glycol to two aldehydes by sodium periodate .
it is possible that intermediate 6 or 9 can be spontaneously converted to the adenine base by amide hydrolysis , 
 or they may remain side products in the reaction rather than formal 
 intermediates . to elucidate the kinetic distributions of the 
 four new species 
 in the alkb reaction , we carried out a series of enzymatic reactions 
 at 37 c with varied time durations at a fixed enzyme 
we chose the time points at 1 , 2 , 5 , 10 , 20 , 40 , 60 , and 80 
 min to quench the reactions and subsequently carried out hplc - esi - tof 
 analysis using an autosampler set to 5 c in an effort to keep 
 the products and intermediates intact .
as presented in figure 4 , we observed that the ea starting material was 
 completely consumed even after the 1 min incubation .
this result indicates 
 that the alkb reaction on ea happened very fast under the current 
 reaction conditions .
we also observed that a major species in all 
 mass spectra was the peak at m / z of 1231.66 ( from 77.4% to 91.5% , figure 4 and table s1 , si ) , which matches up with 
 intermediate with one extra oxygen atom added to ea .
the percentage 
 of this mono - oxidation intermediate decreased as incubation at 37 
 c went to longer times .
the intermediate with two extra oxygen 
 atoms added to ea displayed a relative low percentage for all time 
 periods .
the ratios of the fully repaired product ( undamaged adenine ) 
 and the ea+2 species ( intermediate 6 or 9 in figure 1a ) gradually increased as the 
 incubation time increased ( figure 4 ) . the ratio 
 of adenine increased from 2.6% to 10.0% during the 180 min 
 interval . by performing the hplc - esi - tof analysis of 
 the incubation of ea with alkb using an autosampler at room temperature
, 
 we observed a significantly increased amount of adenine ( figure 3b ) , which might be generated from the spontaneous 
 decomposition of the oxidized intermediates .
percentages ( y - axis ) of the four species in the 
 alkb reactions on ea at 1 , 2 , 5 , 10 , 20 , 40 , 60 , and 80 min incubation 
 time points ( x - axis ) ( see data in table s1 , si ) .
the observed m / z of 1224.73 for the 4 charge 
 monoisotopic peak from the m6a starting material was seen in the absence 
 of alkb ( figure 3c ) . in the presence of the 
 alkb protein ,
the peak at 1224.73 corresponds to the unreacted 
 16mer containing m6a ( about 68% for a 60 min incubation ) .
the 1221.24 
 peak envelope is assigned to the unmodified base adenine ( 15% ) .
the 
 peak envelope at 1228.72 ( 17% ) matches the theoretical mw of the ho - m6a 
 very well ( 1228.71 at 4 charge state ) .
it decomposes to a 
 as the reaction time progresses ( figure s3 , si ) .
we believe this to be the first instance of observing the hydroxymethyl 
 intermediate in the alkb reaction for simple methyl adducts , which 
 strongly supports alkb s oxidative demethylation mechanism . 

with respect to the 100% conversion of m1a under similar reaction 
 conditions , the incomplete repair and buildup of hydroxy intermediate 
 seen for the alkb reaction with m6a suggest that m6a is more sluggishly 
 processed than m1a . unlike the alkb reaction on m6a , we did not observe 
 a hydroxy intermediate ( i.e. , no ho - m1a ) in the m1a reaction .
the 
 different behaviors of m1a and m6a could originate from the charge 
 state of the lesions : at physiological ph , m1a is positively charged , 
 whereas m6a is neutral .
the positively charged ho - m1a intermediate 
 should be less stable than the neutral ho - m6a , leading to faster accumulation 
 of adenine and inability to observe the hydroxy intermediate .
the alleviation 
 of ea toxicity by alkb in e. coli provides 
 an opportunity to investigate further the repair efficiency of such 
 lesions by human homologues , such as habh2 .
if habh2 or its homologues 
 can efficiently repair ea in vivo , the anticancer efficacy of bcnu 
 ( and possibly other dna damaging anticancer drugs ) would be greatly 
 reduced .
the design of a chemical inhibitor to block the repair activity 
 of alkb or habh2 on ea should enhance the anticancer efficacy of bcnu . 

unlike m1a , ea , or ea , the m6a moiety is normally not 
 considered a lesion .
( m5c 
 being the fifth element in addition to the regular four dna bases ) .
the results presented here imply that alkb might 
 not only act as a repair enzyme to defend against exogenous or endogenous 
 attacks by alkylating agents but may also help the cell to control 
 its replication cycle . besides its direct reversal repair of alkyl 
 lesions , such as m1a and ea , alkb , which is strongly induced by dna 
 damage ,
might also demethylate m6a to stop the cell replication cycle 
 temporarily and provide extra time for the cell to repair alkyl adducts . 

after the majority of alkyl lesions are removed , alkb and other repair 
 enzymes can be degraded or down - regulated , followed by progression 
 of the normal cell cycle . the epigenetic control of oxidative demethylation 
 of m6a and its influence on cellular function regulation is also supported 
 by the recent discovery of fto s demethylation of m6a .
in this study , we demonstrated that alkb 
 can demethylate m6a and 
 completely convert ea to undamaged adenine and other intermediates . 

these results indicate that alkb can act not only as a dna repair 
 enzyme but also as a component that may modulate the cellular replication 
 cycle , thus aiding e. coli in surviving 
 alkylation attacks .
further investigation of the cellular functions 
 of alkb will provide insights into alkb s defense mechanisms 
 for the cellular response to environmental attacks as well as the 
 design and development of inhibitors for alkb s human homologues 
 to improve treatments for cancer therapy .
oligonucleotides containing 
 the adducts in figure 1 were prepared using 
 the solid - phase phosphoramidite method , and were deprotected , purified , 
 and identified as described previously .
for all four 
 adducts , the oligonucleotide sequence of the 16mers was 5-gaagacctxggcgtcc-3 , 
 ( x = adduct ) .
dna concentration was 
 measured by uv absorbance using the extinction coefficients (  ) 
 at 260 nm . for any alkyl - modified base
, we substituted its extinction 
 coefficient with the extinction coefficient of its unmodified counterpart 
 due to the tiny difference between the values in the context of a 
 16mer oligonucleotide .
all assays were carried out with alkbn11 protein , a truncated 
 version of alkb in which the first eleven residues were removed .
protein was purified as described and shown previously to have similar 
 activity to the wild type protein . all 
 alkb incubation reactions utilized similar conditions as previously 
 described .
reactions were performed at 37 c in 
 45 mm hepes ( ph 8.0 ) , 0.9 mm -ketoglutarate , 67 m fe(nh4)2(so4)26h2o , and 1.8 mm ascorbate , followed by dry ice storage until hplc - esi - tof 
 ms analysis . a typical reaction was performed with 5 m dna , 
 all cofactors , and with or without 2.5 m alkb in a 10-l 
 volume for 1 h. in the case of a reaction with no alkb protein added , 
 the elution buffer for alkb purification was added instead . for the 
 time and volumetric dependency reactions ( figures 4 in text and s1s4 in si ) , the reaction time and volumetric ratio varied correspondingly .
oligonucleotide analyses were 
 carried out on an agilent esi - tof mass spectrometer ( palo alto , ca ) . 

nitrogen gas was 
 set with drying at 10 l / min , the heated capillary at 325 c and 
 the nebulizer set at 15 psig .
hplc separations were performed by using 
 a zorbax sb - aq column ( 2.1 mm  150 mm ; 3.5 m ; agilent 
 technologies , palo alto , ca ) with a flow rate of 0.2 ml / min .
solvent 
 a used 10 mm ammonium acetate in water ; solvent b used 100% acetonitrile . 

a linear gradient was carried out in the following steps : 2 to 30% 
 b over 30 min , 30 to 2% b over 5 min , and 2 to 2% b over 10 min .
ms 
 analyses were normally carried out at room temperature except for 
 the data presented in figure 4 , which were 
 collected with the temperature of the autosampler set at 5 c . 

 the dna and rna repair protein alkb removes alkyl groups 
 from nucleic 
 acids by a unique iron- and -ketoglutarate - dependent oxidation 
 strategy . 
 when alkylated adenines are used as alkb targets , earlier 
 work suggests that the initial target of oxidation can be the alkyl 
 carbon adjacent to n1 
 . such may be the case with ethano - adenine ( ea ) , 
 a dna adduct formed by an important anticancer drug , bcnu , whereby 
 an initial oxidation would occur at the carbon adjacent to n1 . in 
 a previous study , 
 several intermediates were observed suggesting a 
 pathway involving adduct restructuring to a form that would not hinder 
 replication , which would match biological data showing that alkb almost 
 completely reverses ea toxicity in vivo . 
 the present study uses more 
 sensitive spectroscopic methodology to reveal the complete conversion 
 of ea to adenine ; the nature of observed additional putative intermediates 
 indicates that alkb conducts a second oxidation event in order to 
 release the two - carbon unit completely . 
 the second oxidation event 
 occurs at the exocyclic carbon adjacent to the n6 atom of adenine . 
 the observation of oxidation of a carbon 
 at n6 in ea prompted us to evaluate n6-methyladenine ( m6a ) , an important epigenetic 
 signal for dna replication and many other cellular processes , as an 
 alkb substrate in dna . 
 here we show that m6a is indeed a substrate 
 for alkb and that it is converted to adenine via its 6-hydroxymethyl 
 derivative . 
 the observation that alkb can demethylate m6a in vitro 
 suggests a role for alkb in regulation of important cellular functions 
 in vivo . 
Introduction
Results
Discussion
Conclusions
Experimental Section