Andrés Aguilera
Our laboratory is trying to understand the factors and mechanisms responsible for the origin of genome instability, in particular those leading to recombination and chromosomal rearrangements. In order to maintain stability in the chromosomes, cells contain an array of specific DNA repair pathways that act upon different kinds of lesions, including DNA breaks. A large variety of DNA lesions are produced as a consequence of three main causes: external genotoxic agents such as UV radiation, ionizing radiation and chemical agents causing structural alterations in the DNA and endogenous metabolites such as reactive oxygen species (ROS). All these type of damages can cause cell cycle arrest, replication inhibition or, in pluricellular organisms, cell death. A common and particularly dangereous form of DNA damage is DNA breaks. These can be produced by many different mechanisms, including ionizing radiations, nuclease activity on the DNA or replication fork collapses. They can be repaired by homologous recombination (HR) or Non-Homologous End Joining (NHEJ). A number of experimental evidences relate homologous recombination (HR) to replication in eukaryotes. A preference for HR during S phase relative to G1 has also been shown in chicken, hamster and human cells, and analysis of recombination proteins fused to the green fluorescent protein in yeast has provided evidence that spontaneous recombination foci accumulate during S and G2. The encounter of the replication fork with physical obstacles can be a source of replication stalling and induction of HR. DNA metabolic processes other than replication, and more likely the proteins involved and functioning directly on the DNA, can suppose obstacles for proper replication fork progression that could also demand the assistance of HR. It is becoming evident that recombination DNA repair may be a critical step for DNA replication restart, implying that replication may be a major source of spontaneous double-strand breaks that if not properly repaired can lead to gross chromosomal rearrangements. This is of great importance, given the impact that these types of instability may have on evolution and oncogenesis.

Figure 1: Genome Instability as determined by a colour-assay of recombination in Saccharomyces cerevisiae (left) or by in situ localization of ?-H2AX foci in human cells.
One of our main interests is to understand how endogenous nuclear processes such as transcription, can induce genome instability. There are a large amount of reports indicating that transcription induces hyper-mutation and hyper-recombination. The two phenomena, termed transcription-associated mutation (TAM) and transcription-associated recombination (TAR), may originate from the same transcriptional intermediates. However, they certainly occur by different mechanisms as deduced from the genetic dependence of the different events. It has been recently shown that in the case of TAR, replication may play a mayor role. The collision between the replication fork and the transcription machinery, can lead to a block or collapse of the replication fork, which would require recombination to allow restart. A revealing and unexpected connection between transcription and recombination has been provided by mutants of mRNP assembly/export factors such as THO and others. One likely mechanism to explain TAR is the collision between the transcription and replication machineries. TAR in yeast wild-type cells are linked to impairment of replication fork progression and a connection between blockage of replication forks at natural fob sites in the yeast rDNA regions, transcription and the control of sister-chromatid recombination has added new facts and perspectives for the understanding of TAR. The fact that transcription is coupled with RNA metabolic processes, such as RNA nuclear export, and that defective mRNP biogenesis and export can induce genetic instability bringing new possibilities and mechanisms by which transcription and the whole process of mRNP biogenesis and export can compromise genome integrity by interfering DNA replication.

Our main research goals in this Consolider Programme is to identify and understand the different proteins and mechanisms responsible for genome instability in eukaryotes, in particular those initiated by DNA breaks, transcriptions and obstacles derived from replication fork progression. A large part of our research uses the yeast Saccharomyces cerevisiae, whilst other parts use human and murine cell lines.

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