The mus308 locus of Drosophila melanogaster is implicated in the bypass of ENU-induced O-alkylpyrimidine adducts

Abstract

The mus308 locus of D. melanogaster was originally characterized by virtue of a mutant phenotype that resulted in specific hypersensitivity to cross-linking agents. However, the gene product has also been implicated in the repair of lesions other than cross-links. The gene was recently sequenced, and it encodes a protein with motifs characteristic of both DNA polymerases and helicases. We present mutability studies, using the recessive lethal (RL) test, which show that N-ethyl-N-nitrosourea (ENU) induces hypermutability in mus308-deficient conditions, although only in early broods. Further studies elucidated the role of MUS308 in repair processes by characterizing the spectrum of molecular mutations induced by in vivo ENU in postmeiotic germ cells, in mus308 conditions. These revealed that, in comparison to repair-proficient conditions, there is an increase in the frequency of GC --> AT and AT --> GC transitions, and AT --> TA transversions. Moreover, frameshift mutations, which have not previously been reported to form part of the ENU spectrum, were also found. These results indicate that MUS308 is needed to process ENU-induced lesions, and support the hypothesis that the mus308 gene plays a role in post-replication bypass of O-alkylpyrimidines, probably mediated by recombination, which serves to increase the time available for error-free repair of these persistent and highly mutagenic lesions.

O‐ethylthymidine adducts are the most relevant damages for mutation induced by N‐ethyl‐N‐nitrosourea in female germ cells of Drosophila melanogaster

“…In contrast to E. coli, the alkyltransferases [Sassanfar et al, 1991;Bronstein et al, 1992] and NER machinery [Bronstein et al, 1992] of human cells are relatively deficient in the removal of O 4 -ethylthymine. In Drosophila, O 4 -ethylthymine seems to be partially repaired by NER Tosal et al, 2001] and is a substrate for a bypass-mediated postreplication tolerance mechanism (BMT) represented by the mus308 locus [Tosal et al, 2000]. The results of the present study suggest that this adduct is not repaired efficiently by the O 6 -methylguanine DNA methyltransferase (MGMT) enzyme, which should be active in female germ cells [Vogel, 1989;Dusenbery and Smith, 1996].…”

“…Since this is the premutagenic adduct induced in the greatest concentration by ENU [Singer and Grunberger, 1983;Beranek, 1990], the relatively low frequency of G:C3 A:T transition suggests an efficient repair of this damage, probably by a combination of MGMT [Kooistra et al, 1999;Tosal et al, 1998] and NER [Tosal et al, 2001], as occurs in human cells [Bronstein et al, 1992]. It is possible that these G:C3 A:T transitions could be the result of yet another adduct, like O 2 -ethylcytosine [Jansen et al, 1995;Op het Veld et al, 1997;Tosal et al, 2000], which is a persistent and inefficiently repaired lesion [Brent et al, 1988] induced by ENU in relatively high concentrations [Beranek, 1990]. The observation that G:C3 A:T transitions were isolated only in mature oocytes, where DNA repair has little time to remove damage, suggests that these mutations result from O 6 -ethylguanine adducts.…”

“…In mammals, although some results point to a possible role for NER in the repair of O 2 -ethylthymine [Op het Veld et al, 1997], there are other data indicating that this system does not participate in the repair of this damage [Brent et al, 1988;Bronstein et al, 1992]. In D. melanogaster, O 2 -ethylthymine is a substrate of the BMT system [Tosal et al, 2000], but there is no proof for its repair by NER [Tosal et al, 2001].…”

“…Genomic DNA was isolated from about 1 g of flies, according to Pastink et al [1989]; 0.2 g of this genomic DNA were used to amplify the vermilion gene by polymerase chain reaction (PCR) [Saiki et al, 1985;, according to the method of Tosal et al [2000]: 100 l of reaction contained 30 pmoles of each primer, p.␣ (5Ј-CGCCCAGATCACCGACT-GTGC-3Ј) and p. (5Ј-GCTGAAATTAATGGTGCGAATTTGG-3Ј), 10 l of 10 ϫ amplification buffer (0.1 M Tris-HCl pH 8.4; 20 mM EDTA; 0.1 M NaCl), and 0.2 mM of each deoxynucleotide triphosphate. The amplification conditions were: 1 min at 95°C; 30 cycles of 30 sec at 95°C, 1 min at 60°C, 1 min at 72°C;, followed by 5 min at 72°C.…”

Recombinational Repair Genes and Breast Cancer Risk

“…However, it has been reported that mus308 might be also involved in postrepUcational repair (AGUIRREZABALAGA rt aZ. 1995;TosAL et al 2000). Homozygous mus308 flies showed elevated embryonic mortality associated with chromosome instability and a mutator phenotype in response to certain mutagens (LEONHARDT et al 1993).…”