DNA double strand breaks occur spontaneously or as a result of DNA damaging agents such as ionizing radiations or chemical reagents. If this damage is not properly repaired, it can lead to the occurrence of chromosomal rearrangements such as duplications, deletions and translocations, which can affect cell growth and survival. These rearrangements are key events in genome reshaping and evolution processes and many of the genomes sequenced to date show traces of these rearrangements [1
]. In multicellular organisms, however, chromosomal rearrangements are often responsible for oncogenesis and for many human genetic diseases [3
DNA double-strand break (DSB) repair mechanisms are therefore essential to each organism, since they preserve the integrity of the genome and prevent the deleterious effects of chromosomal rearrangements. These mechanisms can be classified in two distinct pathways: the homologous recombination (HR) pathway and the non-homologous end-joining (NHEJ) pathway. HR requires long homologous sequences for DSB repair whereas little or no homology is necessary for the NHEJ pathway.
In order to select spontaneous chromosomal rearrangements, a genetic screening method based on a particular allele of the URA2
gene was developed (Figure ) [6
]. The URA2
gene is located on chromosome X and encodes a multifunctional protein, catalyzing the two first steps of the pyrimidine biosynthesis pathway and composed of glutamine amidotransferase (GATase), carbamoylphosphate synthetase (CPSase) and aspartyltranscarbamylase (ATCase) domains. The ura215,30,72
allele has three point mutations located in its proximal region, which result in the loss of all the activities encoded by the URA2
gene. Both CPSase and GATase activities are compensated by two isoenzymes of the arginine biosynthesis pathway whereas the ATCase activity isn't. Thus the ura215,30,72
strain is auxotrophic for uracil. However the ATCase activity can be reactivated by complex chromosomal rearrangements. This powerful screening tool can be used to perform in vivo
experiments without the side-effects observed when using mutagenic agents or plasmid-encoded reporter genes.
Figure 1 The ura215,30,72 allele and its corresponding multifunctional protein Ura2p. ns15 and ns30 correspond to the positions of the non-sense mutations and fs72 to the position of the frameshift mutation. GATase stands for Glutamine AmidoTransferase, CPSase (more ...)
In previous studies, rearrangements of several kinds were observed using this URA2
-based screening method [6
]. In the haploid FL100 context, three types of rearrangements leading to ATCase reactivation were detected: Ty1 insertions downstream of the last point mutation in the ura215,30,72
allele, deletions of the region containing the three mutations, and duplications of the region encoding the ATCase followed by fusion with a new promoter sequence. Roelants et al.
studied the ATCase reactivation resulting from Ty1 insertions and established that the transcription process is initiated in the LTR (Long Terminal Repeat) region of the Ty1 retrotransposon [8
]. Deletions of the mutated region in the ura215,30,72
allele were described by Welcker et al.
and duplications of the ATCase region by Schacherer et al.
An analysis of variations at the nucleotide level in some commonly used Saccharomyces cerevisiae
strains was carried out by Schacherer et al.
, who detected SNPs (Single Nucleotide Polymorphism) and deletions in various strains [10
]. These sequence differences may have important effects on several biological pathways and phenotypes. A total number of 22,446 SNPs and 53 deletions were identified when the FL100 strain was compared to the S288c strain and the divergence observed between the two strains amounted to 0.21% [10
]. To assess the effects of the genetic background on mutation rates and the type of chromosomal rearrangements, the ura215,30,72
genetic screening was used to select spontaneous rearrangements in the S288c context. The results were compared with those previously obtained in the FL100 background [6
Interestingly, in the S288c background, while duplications and deletions events were found to be responsible for the ATCase reactivation, no Ty1 insertions were observed. It was therefore concluded that the occurrence of chromosomal rearrangements is background-dependent. In addition, the occurrence of chromosomal deletions and duplications due to various recombination mechanisms was studied in haploid contexts. The impact of homologous recombination was tested by selecting revertants in a Δrad52 strain. Since Rad59p plays an important role in single-strand annealing (SSA) processes between direct repeats, a Rad59p deficient mutant, Δrad59, was constructed and selections of chromosomal rearrangements were performed in this background. The mutation rates and types of rearrangements observed in these two contexts were compared with a reference ura215,30,72 strain. Inhibiting RAD52-dependent homologous recombination increased the deletion rate, whereas inactivation of the SSA pathway increased the duplication rate. Secondly, the effects of non-homologous end joining (NHEJ) were tackled by mutating LIG4 and YKU80 in the ura215,30,72 background. The LIG4 mutation affected the mutation rates for both deletions and duplications, however there was no effect observed with YKU80 deletion. These results lead us to conclude that a Yku80p-independent NHEJ mechanism is responsible for the occurrence of chromosomal deletions and duplications.