among the genes identified so far in drosophila melanogaster that play a role in dna damage repair , mus308 presents some unique properties , because its cdna sequence shows motifs characteristic of dna helicase and dna polymerases . the putative product of this gene was indeed isolated as a new dna polymerase , homologue to the escherichia coli dna polymerase i , carrying as well a dna helicase domain at the n terminus region .
orthologues of this gene have been found in caenorhabditis elegans , arabidopsis thaliana [ 3 , 4 ] , and mammals [ 3 , 59 ] . in humans ,
three genes encoding proteins with sequence similarities to mus308one similar to mus308 helicase , hel308 , and two similar to the mus308 polymerase , polq [ 5 , 7 ] and poln have been identified to date .
polq , the most studied of these proteins , has also an atpasehelicase domain at the n terminus and is able to perform dna synthesis past an abasic site , following the a - rule ; however , there are contradictory results about its fidelity in a normal nondamaged template [ 10 , 11 ] .  
the mus308 gene is involved in the repair of cross - linking adducts [ 12 , 13 ] and also of monofunctional damage , probably persistent and difficult to repair by other systems , such as the o - ethylpyrimidine damage induced by n - ethyl - n - nitrosourea ( enu ) in postmeiotic male germ cells .
in addition , at least parts of enu- and diethyl sulphate- ( des- ) induced damages were repaired by mus308 in female germ cells of drosophila .
this protein works in a damage bypass mechanism [ 1 , 13 ] , which was originally related to homologous recombination , hr [ 14 , 16 ] .
nevertheless , the isolation of the dna polymerase encoded by this locus , its possible ability for dna synthesis through abasic sites [ 10 , 11 ] , and the requirement of a functional mus308 protein to prevent damage - induced dna strand breaks in vivo in somatic cells of drosophila , pointing to a translesion synthesis ( tls ) mechanism as the activity of this protein . in summary , along these years the work of our laboratory have demonstrated that mus308 works in the repair / processing of cross - links and oxygen ethylation damage [ 1315 , 17 ] whereas n - ethylation damage is apparently not substrate of this system , because no effect of methyl methanesulphonate ( mms ) was detected either in germ cells   or in somatic ones .
additionally , its mechanism of action is poorly understood , because it could be related to hr [ 14 , 16 ] or to tsl .
because of this , we have proposed that the mus308 locus works in a bypass - mediated tolerance mechanism , btm [ 15 , 17 ] .    given the conservation of mus308 among higher eukaryotic organisms , this locus is likely a part of a repair system relevant to dna damage processing .
therefore , it would be important to elucidate what types of dna damage , apart from cross - links and o - ethylpyrimidine adducts , are substrate of this system and to get information about which of the two possible mechanisms of action , hr or tls , is actually involved in the damage bypass process . to have more information about the role of mus308 in the processing of dna ethylation damage , we have studied here the effect of des in postmeiotic male germ cells , analysing maternal repair and using the vermilion system . this system combines the analysis of induced mutation frequencies , both at a single locus ( vermilion , with a specific locus test ) and at multiloci ( 700 loci in the x - chromosome , with the recessive lethal test ) , with the generation and analysis of mutation spectra .
our data , together with other already published , indicate that mus308 protein is involved in the processing of all types of oxygen ethylation damage , that it is also involved in the processing of nitrogen ethylation damage , that it prevents cell death at least when the amount of dna damage is high , and that this protein could be working in a tls mechanism as well as in an alternative end - joining system ( alt - ej ) .
6467 - 5 ) , obtained from sigma qumica ( spain ) , was dissolved in a solution of 3% ethanol-1% tween-80 in 33.1  mm phosphate buffer ( 16.5  mm na2hpo4 , 16.6  mm kh2po4 , ph 6.8 ) , containing 5% sucrose .
1 - 2-days old brown ( bw ) males , in groups of 30 individuals , were placed in glass tubes , with eight layers of glass microfiber paper ( whatmann , gf / a ) at the bottom , soaked with 0.9  ml of different des concentrations .
after 3 hours treatment , males were mated to in(1)sc^s1l sc^8r in(1)dl" " 49 , y , scsc , v ; bw ; mus308 ( i , v ; bw ; mus308 ) virgin females ( for marker descriptions see ) .
protocols for fractionation the progeny in mature sperm and spermatids for the recessive lethal ( rl ) test and for isolation of f1 and f2vermilion ( v ) mutants were described elsewhere .  
at least five different experiments were carried out for each concentration and , since there were no differences among them , data were pooled .
statistical analysis of rl results was performed comparing mutant frequencies in treated flies with their respective negative controls , using the fisher exact test .  
the influence of mus308 in the repair or processing of ethylation - induced damage was measured through the mutability index ( mi ) , and the statistical analysis of aguirrezabalaga et al .  
for each transmissible vermilion mutant , a homozygous strain was established to carry out the molecular analysis .
all the isolated mutants were analysed . the isolation of dna and pcr amplifications were as described .
sequencing reactions for the coding region were carried out using the dideoxy method , with a set of 10 internal primers . a fragment of about 1.8  kb , localized upstream of the coding region ,
was analyzed as described before   in those mutants which did not show changes in the coding sequence . in order to exclude taq polymerase - introduced errors , at least two plaques or colonies from independent pcr reactions
statistical analyses of differences between mutation spectra were carried out using the hypergeometric test for comparison of samples from mutational spectra [ 23 , 24 ] .
the rl and vermilion mutation frequencies , both spontaneous and induced by the different des concentrations under mus308 deficient ( mus308 ) conditions , are presented in table 1 .
pooled data from mature sperm and spermatids are shown , because no differences between them were found in any case ( not shown ) .  
all chemically induced rl mutation frequencies are statistically higher than the spontaneous one , although their values decrease as des concentration increases .
comparisons with the results previously obtained in mus308 proficient ( mus308 ) conditions   reveal two relevant differences ( table 1 ) .
first , the spontaneous rl frequency is statistically lower in mus308 than in mus308 conditions .
second , in mus308 conditions , a decrease in rl frequencies is induced as des concentration increases , whereas the opposite , that is an increase was detected in efficient repair conditions .
consequently , the value of the mutability index ( mi ) for 10  mm des is statistically higher than 1 whereas for 15  mm and 25  mm the mis are lower than 1 ( table 1 ) . to analyse the dose range between 10 and 15  mm des , and to compare both repair conditions in the same experiment ,
the obtained results confirmed that when the amount of dna damage is low or moderate , hypermutability is obtained ( 4.8% and 8.2% rl mutation frequencies in mus308 and in mus308 , respectively , with a statistically significant mi of 1.7 ) .
it is noticeable that the very high rise in mutation frequency was detected between 1012  mm and 15  mm des in efficient repair conditions , but it is not unusual to find such a narrow window of increased activity in a chemical .
the obtained results demonstrate that mus308 detects and processes des - induced dna damages . on one hand , low effectiveness des doses , such as 1012  mm ( inducing low mutation frequencies ) ,
cause dna damage , mostly oxygen alkylations , that seems to be processed through an error - free pathway , as pointed by the observed hypermutability . on the other hand , with high effectiveness
des doses ( such as 15 and 25  mm ) , able to induce also considerable nitrogen alkylations , the obtained results indicate hypomutability ; this fact , together with a decreased induced fertility , suggest that a functional mus308 protein is necessary for the survival of the fertilized eggs .
the analysis of vermilion mutation frequencies ( table 1 ) show that , under mus308 conditions and considering all concentrations together , 2 mutants were isolated in f1 ( 0.11  10 mutation frequency ) while most v mutants , 11 , were isolated among the f2 offspring ( 1.46  10 mutation frequency ) .
other 5 v mutants were isolated from mass cultures , but they are not included in the mutation frequency estimations .
additionally , another mutant induced by des was identified by genetic analysis as a translocation between the x and y chromosomes that does not include the v locus .
a comparison of these data with those obtained under mus308 proficient conditions   reveals that the f1 and f2 induced mutation frequencies are much lower in mus308 than in mus308 conditions , indicating that the hypomutability observed with rl frequencies also extends to v mutation frequencies .  
these results are in agreement with those previously obtained with enu in the same cell type and under the same repair conditions . in that case ,
hypermutability was observed with a concentration that induced a moderate level of dna damage ( 1  mm enu ) , and similar results were found in rl and vermilion mutation frequencies analysis .    
results obtained here are consistent with a hr - mediated bypass of dna damage if at least part of this damage induces cell mortality .
however , a bypass tolerance system mediated by tls could be also implicated in the processing of des induced damaged .
thus , a dna polymerase could process error - free some dna damage , like oxygen alkylation , when the amount of dna damage is low , but the processing of other types of induced dna lesions , especially when they are present in high amounts ( because other repair systems are saturated or inactive ) , like nitrogen alkylation , could be error - prone .  
moreover , there is another tolerance system , the alternative end - joining process ( alt - ej ) , independent of ligase 4 .
mus308 was very recently discovered to be involved in this system , which processes dna double strand breaks generated by replication blockage .
our results are compatible also with this system because nitrogen alkylations can be the source of dna strand breaks .
details of des - induced mutants are shown in table 2 . in d8 - 9
no mutation was found , and the same mutation was present in the independent mutants d8 - 21 and d8 - 30 , as previously reported for other vermilion spectra [ 14 , 18 , 21 , 31 , 32 ] . in d8 - 26
no mutation was detected in the coding region nor in the proximal part of the promoter , but the distal part of the promoter could not be amplified , suggesting the presence of a mutation .
additionally , d8 - 6 presented five different mutations none of which was found in any other mutant .
no mutants were isolated either from the 24002 f1 and 15698 f2 flies analysed in the concurrent control experiments or in the historical control ; therefore , we consider that the observed mutations were induced by des .
the v mutation spectrum , constituted by the 23 obtained mutations , is summarized in table 3 and includes two deletions ( 8.7% ) and 21 base pair changes , distributed as follows : 11 gc - at ( 47.8% ) and 4 at - gc ( 17.4% ) transitions , and 4 at - ta ( 17.4% ) and 2 gc - ta ( 8.7% ) transversions .  
the pairing up of these mutations with the several adducts induced by des indicates that : ( i ) the gc - at and at - gc transitions in the des spectrum should be , respectively , the consequence of the o - ethylguanine and o - ethylthymine adducts [ 3336 ] , induced by this chemical [ 3739 ] ; ( ii ) at - ta transversions are most probably due to n - ethylation , like the rest of transversions and the deletions [ 25 , 27 ] , because des does not ethylate o - thymine .
the two found deletions occur between direct repeats and , as the translocation , they can be indirectly generated from n - ethylation , as described before .
comparison of the relative mutation frequencies of this mus308 spectrum with those previously obtained under mus308 conditions ( table 3 ) reveals clear differences ( p = .07 , with the hypergeometric test , and lower if the translocation is considered ) , including a strong decrease in the frequency of gc - at transitions , and increases in the frequencies of at - gc transitions , transversions and deletions under mus308   conditions .
these results confirm that mus308 is processing o - ethylguanine and o - ethylthymine , as indicated before , and reveal that this protein is also processing n - ethylation damage .  
o - ethylguanine , like o - methylguanine , is a stable dna lesion   that can mispair with t to produce gc - at transitions as indicated but can also pair correctly with c   or can even block dna polymerases [ 41 , 42 ] .
o - ethylthymine is a dna damage with a long half - life , difficult to repair in mammals [ 4345 ] .
although it is not considered a lethal lesion [ 33 , 36 ] , it is able to block dna replication in mammalian cells in a ner deficient background .
ner is apparently implicated in its repair in drosophila [ 27 , 32 ] , although rather inefficiently , because at - gc transitions are one of the most frequently enu - induced damages in the repair - active premeiotic germ cells of this organism [ 21 , 31 ] .
therefore , this adduct fits with the proposed requirements for the substrates of mus308 [ 13 , 14 ] .
since at least part of the n - ethylation damage can be persistent and can block dna synthesis , its detection as substrate of mus308 , especially when the level of dna damage is very high and repair is difficult , is not unexpected or strange .
additionally , it can be considered that n - ethylation is a source of dna strand breaks , and this type of dna damage is substrate of mus308 in the alt - ej system .
the sequence specificity of des- induced mutations was studied determining the base pairs 5 and 3 of the damaged nucleotide ( table 2 ) .
the results of this analysis show that at pairs are present at 5 in 64% of gc - at mutations in mus308 conditions whereas 64%70% of this type of mutations is preceded by gc pairs at 5 in mus308 conditions   and in ner deficient conditions , respectively .
this means that the neighbouring sequences 5 to o - ethylguanine change depending on the mus308 status , which is in good accordance with the proposed polymerase function of mus308 , specially considering that no influence of surrounding sequences was found before for this chemical in this locus [ 25 , 27 ] , nor were expected for an sn1/sn2 alkylating agent . in summary ,
the results presented in this paper demonstrate that mus308 processes oxygen and nitrogen alkylations , and they support its role in a tolerance mechanism that is especially relevant in case of high dna damage levels , because it prevents cell death .
additionally , these results suggest that this protein could act through a tls pathway , because of ( i ) the detected neighbouring sequence influence , and ( ii ) its dna polymerase activity .
finally , these results also agree with the mus308 role in the alt - ej system , for the processing of dna damage - inducing strand breaks , which can be compatible with the tls pathway .
 the d. melanogaster mus308 gene , highly conserved among higher eukaryotes , is implicated in the repair of cross - links and of o - ethylpyrimidine dna damage , working in a dna damage tolerance mechanism . however , despite its relevance , its possible role on the processing of different dna ethylation damages is not clear . to obtain data on mutation frequency and on mutation spectra in mus308 deficient ( mus308 ) conditions , the ethylating agent diethyl sulfate ( des ) 
 was analysed in postmeiotic male germ cells . 
 these data were compared with those corresponding to mus308 efficient conditions . 
 our results indicate that mus308 is necessary for the processing of oxygen and n - ethylation damage , for the survival of fertilized eggs depending on the level of induced dna damage , and for an influence of the dna damage neighbouring sequence . 
 these results support the role of mus308 in a tolerance mechanism linked to a translesion synthesis pathway and also to the alternative end - joinig system . 
1. Introduction
2. Material and Methods
3. Results and Discussion