DNA carries all the genetic information for life and thus must be duplicated faithfully and transferred to each new cell during cell division. DNA duplication is achieved by the intrinsic high fidelity of the eukaryotic DNA replication machinery. However, imperfections in the DNA template or physical roadblocks can threaten the fidelity of the replication process and thereby jeopardize genomic integrity. It is therefore essential that the cell is able to cope properly with roadblock events. Failures in maintaining genome stability inevitably result in the accumulation of mutations, genome rearrangments, cell death and cancer. We study cellular pathways that protect the stability of eukaryotic chromosomes upon replication roadblocks.
We take out-set in two unique cellular system that recently were developed in the lab and which allow induction of roadblocks at well-defined genomic places. They thus form the basis of our research aiming to understand the interplay between cellular pathways, which guards the genome upon replication fork stalling. These pathways include among others, checkpoint, repair and replisome integrity pathways.
The Fob-block system: This system is engineered to facilitate studies of replication fork stalling at a physical non-covalent protein-DNA roadblock. Replication fork barrier (RFBs) sequences have been inserted ectopically on chromosome VI in non-transcribed regions. RFBs are highly conserved in eukaryotes and naturally present in the rDNA, where they generate unidirectional replication as they inhibit leftward moving replication forks. The exact mechanism of RFB function is unknown, but depends on the tight binding of the Fob1 protein to the RFB sequence, which is thought to warp around the protein. To obtain the controllable Fob-block system, the FOB1 gene is placed under the control of the inducible GAL1/10 promoter. Thus, when cells are grown in presence of galactose, the protein-DNA barriers are activated which will stall replication forks. Growth in the presence of glucose will suppress Fob1p expression and the protein-barrier is therefore not activated (see movie 1).
The Flp-nick system: This system is engineered to investigate the consequences of replication fork collision with a protein-associated DNA nick. This mimics the type of roadblock generated by DNA topoisomerase I (Top1), which is a ubiquitous enzyme that regulates DNA topology by relaxing positive and negative supercoiling in the DNA that arise during replication and transcription. The Flp-nick system takes advantage of the site-specific Flp recombinase found in S. cerevisiae, which is related to Top1 and executes the same catalytic mechanism. Flp recombinase cleaves at well-defined sequences (Flp recognition target, FRT), which makes it a great tool in generating site-specific roadblocks. The Flp-nick system utilizes an Flp mutant, which will generate a permanent protein-associated DNA, nick upon cleavage. A Flp recognition target (FRT) has been inserted in the genome of S. cerevisiae and cells express the Flp recombinase mutant behind the inducible GAL1/10 promoter, which allows for controlled expression of the enzyme (see movie 2).
Techniques applied in the lab: High throughput genomic screens to identify new players essential for roadblock survival, chromatin immunoprecipitation, real time PCR, replication 2D-gels, puls field gel electrophoresis, fluorescent microscopy, fluorescence activated cell sorting, specific repair and checkpoint assays, traditional molecular biology techniques and yeast genetics.