Effect of nucleotide excision repair on ENU‐induced mutation in female germ cells of Drosophila melanogaster

Abstract

The role of nucleotide excision repair (NER) in the repair of alkylation damage in the germ cells of higher eukaryotes has been studied mainly by treating postmeiotic male germ cells. Little is known about repair in actively repairing female germ cells. In this study, we treated NER-deficient (ner(-)) mus201(D1) Drosophila females with N-ethyl-N-nitrosourea (ENU) and determined both the mutant frequencies in the multiple locus recessive lethal (RL) test and in the single locus vermilion gene and determined the ENU mutation spectrum in the vermilion gene. The results show that ENU is mutagenic in all cell stages and that the induced frequencies increase with cell maturation, from oogonia to mature oocytes. In addition, the induced spectrum consists mainly of A:T-->T:A transversions (43.8%), A:T-->G:C transitions (21.9%), and A:T-->C:G transversions (15.6%). G:C-->A:T (3.1%) transitions, other transversions (9.4%), frameshifts (3.1%), and deletions (3.1%) were also found. Comparison of these results with those previously obtained for repair-proficient (ner(+)) female germ cells reveal: 1) Differences in the RL and vermilion mutation frequencies for ner(+) and ner(-) germ cells, indicating that NER is involved in the repair of ENU-induced damage to these cells. 2) At least 15.6% of mutations in ner(-) cells may be the consequence of N-ethylation damage and mutations of this type were not detected in ner(+) cells. 3) Although differences were found in transition frequencies between ENU-treated ner(+) and ner(-) germ cells (52.2% vs. 25%), suggesting that a functional NER is involved in processing O-ethylated damage, the role of NER in repairing O-ethylated adducts is uncertain.

Poly (ADP‐ribose) polymerase‐1 deficiency does not affect ethylnitrosourea mutagenicity in liver and testis of lacZ transgenic mice

“…Although this hypothesis is compatible with our present data, in that no effect of Parp1deficiency was detected in ENU-induced mutations, it cannot explain the previous results with MNU that showed an accumulation of large deletions/insertions in the absence of Parp1 activity [Louro et al, 2008]. This discrepancy may be explained by BER becoming inefficient when Parp1 is abrogated and other Parp1-independent repair mechanism becoming operative in the removal [Pegg and Byers, 1992;Op het Veld et al, 1997;Á lvarez et al, 2003] and may also repair N-ethylated bases upon BER impairment as was suggested in studies with Drosophila melanogaster [Á lvarez et al, 2003]. In contrast, MNU adducts are not substrates for NER and the great majority of the premutagenic MNU-DNA adducts (especially O 6 -methylguanine) that are not directly repaired by alkylguanine transferases are removed from DNA exclusively by the BER pathway [Op het Veld et al, 1997].…”

“…DOI 10.1002/em of N-ethylated purines but relatively inefficient in the repair of MNU-DNA adducts. Supporting this idea, it is known that NER participates in the repair of O 6 -ethylguanines and O 2 -ethylthymines [Pegg and Byers, 1992;Op het Veld et al, 1997;Á lvarez et al, 2003] and may also repair N-ethylated bases upon BER impairment as was suggested in studies with Drosophila melanogaster [Á lvarez et al, 2003]. In contrast, MNU adducts are not substrates for NER and the great majority of the premutagenic MNU-DNA adducts (especially O 6 -methylguanine) that are not directly repaired by alkylguanine transferases are removed from DNA exclusively by the BER pathway [Op het Veld et al, 1997].…”

Mus308 Processes Oxygen and Nitrogen Ethylation DNA Damage in Germ Cells of Drosophila

“…Mutant vermilion genes were cloned in the M13mp19 vector or in a pUC18 plasmid [22]. Sequencing reactions for the coding region were carried out using the dideoxy method, with a set of 10 internal primers.…”

Machine learning techniques for classifying the mutagenic origins of point mutations

“…The systematic use of mutagens in forward genetic screens provides an opportunity to develop an understanding of the relationship between neighboring sequence and mutagenesis. N-ethyl-N-nitrosourea (ENU) is a synthetic alkylating chemical widely employed in mutagenesis studies (Álvarez et al, 2003;Lee et al, 2012;Stottmann and Beier, 2014), causing new germline mutations at ∼100 times higher rate than the spontaneous mutation rate (Stottmann and Beier, 2014). Exposure to ENU can induce formation of a number of alkylation adducts including N 1adenine (e 1 A), O 4 -thymine (e 4 T), O 2 -thymine (e 2 T), and O 2 -cytosine (e 2 C) (Shrivastav et al, 2010;Noveroske et al, 2000).…”