Abstract
While in mammalian cells the genetic determinism of chromosomal translocation remains unclear, the yeast Saccharomyces cerevisiae has become an ideal model system to generate ad hoc translocations and analyze their cellular and molecular outcome. A linear DNA cassette carrying a selectable marker flanked by perfect homologies to two chromosomes triggers a bridge-induced translocation (BIT) in budding yeast, with variable efficiency. A postulated two-step process to produce BIT translocants is based on the cooperation between the Homologous Recombination System (HRS) and Break-Induced Replication (BIR); however, a clear indication of the molecular factors underlying the genetic mechanism is still missing. In this work we provide evidence that BIT translocation is elicited by the Rad54 helicase and completed by a Pol32-independent replication pathway. Our results demonstrate also that Rdh54 is involved in the stability of the translocants, suggesting a mitotic role in chromosome pairing and segregation. Moreover, when RAD54 is over-expressed, an ensemble of secondary rearrangements between repeated DNA tracts arise after the initial translocation event, leading to severe aneuploidy with loss of genetic material, which prompts the identification of fragile sites within the yeast genome.
Highlights
Chromosomal translocations characterize many types of cancers [1,2] and can become useful markers in the diagnosis of liquid and solid tumors [3,4]
We showed that transformation with a bridge-induced translocation (BIT) cassette harboring two homologies for two distinct chromosomes of yeast generates an ensemble of events that are completely different from those observed in a GT experiment and probably are due to activation of different molecular pathways
Rad54 is a member of the SWI/SNF-chromatin remodeling complex and of the Rad52-epistasis group in which it is essential for the turnover of Rad51 at the double strand break (DSB) and, it is responsible for the transition from homologous pairing to DNA synthesis [41]
Summary
Chromosomal translocations characterize many types of cancers [1,2] and can become useful markers in the diagnosis of liquid and solid tumors [3,4]. The method, which exploits the homologous recombination system of yeast, consists of the DNA transformation of diploid cells with a PCR-amplified selectable DNA fragment carrying at its ends sequences homologous to the chromosomal sites chosen for translocation. We discovered that this kind of bridge-induced translocation is non-reciprocal, that its frequency depends on the extension of the homology [8], that it occurs with similar efficiency between heterologous and homologous chromosomes [9] and that it is usually associated with the formation of viable aneuploids [10].
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