RESEARCH PROJECTS
Wolf-Dietrich Heyer

1. Mechanism of homologous recombination in eukaryotes

Several pathways ensure the repair of DSBs in eukaryotic cells. Recombination is the only inherently error-free pathway to accomplish this and it involves a complex series of events which are now beginning to be understood in eukaryotes (see Figure 1). Other pathways like Non-Homologous Endjoining (NHEJ) and Break-Induced Replication (BIR) are error-prone, leading to mutations and loss of heterology. These DSB repair pathways are conserved in all eukaryotes. In fact, recombinational repair is conserved in all forms of life. Recent progress in identifying the components of recombinational repair has provided convincing evidence that this entire pathway is conserved. Recent work with transgenic mice has also provided conclusive evidence that recombinational repair is active and important in mammalian cells. Interestingly, this pathway is also crucial for the repair of DNA crosslinking agents, another class of anti-cancer drugs. Purification of the individual components of this DSB repair pathway and defining the biochemical function of the proteins, alone and in conjunction, is crucial to understand the mechanism of recombinational repair.

Based on the premise of evolutionary conservation of the DNA repair pathways, it appears that the basic mechanism of DSB repair will be highly similar in all eukaryotes. The situation in more complex eukaryotes like mammals will undoubtedly be more sophisticated in details. Thus, we have decided to concentrate the mechanistic studies on the yeast S. cerevisiae model system, where many components are identified and genetic analysis is easily possible.

Our mechanistic work started with Rad54 protein, one of the most crucial DSB repair proteins (Clever et al. 1997; Schmuckli-Maurer and Heyer 1999; Clever et al. 1999; Schmuckli-Maurer and Heyer 2000; ). The final goal is the biochemical reconstitution of the entire pathways including the following proteins: Rpa, Rad51, Rad52, Rad54, and Rad55/57.

Presently, we have developed in vitro recombination reactions with Rpa, Rad51, and Rad54 proteins (Mazin et al. 2000; Solinger et al. 2001; Solinger and Heyer 2001). Before adding more complexity to this system, we want to elucidate the exact function of the Rad54 protein. Rad54 protein interacts specifically with Rad51 protein in the presynaptic filament to stimulate homologous pairing and DNA strand exchange in the synaptic (Mazin et al. 2000; Solinger et al. 2001) and post-synaptic phases (Solinger and Heyer 2001) of recombination. We are currently working on understanding the mechanism by which Rad54 protein stimulates Rad51 protein-driven DNA strand exchange. Our efforts are focused on the role of the dsDNA-dependent ATPase activity of Rad54 (Kiianitsa et al. 2002), and we demonstrated that Rad54 can disassemble Rad51:dsDNA filaments (Solinger et al. 2002). Our present working model, which is consistent with all genetic and cytological evidence that suggests a role of rad54 after Rad51/Rad52/Rad55/57, suggests that Rad54 turns over of the Rad51 product DNA complex, when Rad51 is bound to the hybrid DNA after DNA strand exchange to control access to the 3'-OH of the invading strand by DNA polymerases.

In addition, we have identified a significant increase in genomic instability (chromosome loss) in Saccharomyces cerevisiae cells lacking Rad54 protein and are conducting experiments the identify the mechanisms that contribute to genomic instability in rad54 mutants (Schmuckli-Maurer et al. 2003).

Mus81/Mms4 is a DNA structure-specific endonuclease that we identified in a two hybrid screen using Rad54 as a bait (Interthal et al. 2000). Further genetic analysis provides strong arguments that Mus81/Mms4 functions late in recombination in a resolution pathway that is parallel to Sgs1/Top3 (Fabre et al. 2002). While the exact function of Mus81/Mms4 in recombination is unclear, it appears likely that it is improtant for the recovery of stalled and/or broken replication forks (Heyer et al. 2003).

2. Regulation of DSB repair by DNA damage checkpoints

DNA damage checkpoints coordinate the cellular responses to DNA damage including transient cell cycle arrest and replication slow down, transcriptional induction of a large array of genes, and - in higher cells - programmed cell death (see Figure 2). Defects in this signal transduction pathway leads to major radiosensitivity in all organisms studied and to hereditary cancer predisposition in humans, as exemplified by the syndrome ataxia telangiectasia (AT). A growing number of components of this pathway has been isolated, primarily through efforts in yeast model systems. However, the mechanism how the checkpoints recognize DNA damage and elicit cellular responses is almost completely unknown. Seminal work with cells derived from AT patients suggested DNA repair defects in such cells that did not eliminate but somehow misguide DNA repair of DSBs.

Figure 2. DNA damage and replication block checkpoints in S. cerevisiae and the functions of checkpoint proteins.

We recently entered the checkpoint field based on the premise that the DNA damage sensing capability of checkpoints might provide a direct way for the DNA repair systems to be recruited to the sites of DNA damage. In particular the biochemical properties of the DSB recombinational repair pathway suggested that DNA damage recognition is a major problem. Thus, we have directly analyzed the components of this pathway if they are substrates for the DNA damage checkpoints. We have identified that Rad55 protein is specifically phosphorylated in response to variety of DNA damages including ionizing radiation (even a single DSB) dependent on an active checkpoint (Bashkirov et al. 2000). Thus we have established that both systems, checkpoint control and DNA repair are directly linked. Currently we are putting much effort in identifying the Rad55 protein kinase, to establish the biological significance of this phosphorylation, and the functional difference between phosphorylated and unphosphorylated Rad55 protein.

Rad55 phosphorylation is a terminal checkpoint event and provides a unique opportunity to analyze the regulation of the upstream signal transudction cascade. In trying to understand how the checkpoint kinases are regulated, we demonstrated that Dun1 kinase is directly phosphorylated by Rad53 kinase after genotoxic stress (Bashkirov et al. 2003). The specificity of this interaction is determined by the FHA domain of Dun1. The interaction between activated Rad53 kinase and Dun1 is highly transient and destabilized by autophosphorylation of Dun1 after Dun1 is activated by trans-phosphorylation by Rad53. This provides a mechanism for signal amplification, as one activated Rad53 kinase moleculae can potentially activate many Dun1 kinase molecules. These studies also revealed a Rad53-independent role of Dun1 kinase, which we would like to understand.

3. Identification and characterization of novel DNA repair genes and their human counterparts

The determination of the entire genome sequence of S. cerevisiae and the nearing completion of the fission yeast sequence allows novel, genome-wide approaches to identify novel DNA repair genes. As part of the EUROFAN program, we have embarked in coordination with other laboratories to identify novel DNA repair genes among the orphan open reading frames of the budding yeast genome. This program not only identifies novel genes functioning in the mechanisms and regulation of DNA repair, it also provides the basis to isolate such genes from mammals including humans. The growing human databases allow direct analysis for homologs using biocomputing tools. In addition, the cross-comparison between the distantly related budding yeast and fission yeast will identify genes that are conserved in both organisms and likely in all eukaryotes. This allows direct cloning strategies like degenerate PCR. Moreover, mutant yeast strains provide the perfect laboratory to study the function of mammalian repair genes.

In collaboration with Drs. Kanaar and Hoeijmakers (Erasmus University, Rotterdam) we have shown that the human Rad54 cDNA can partially rescue the DNA repair defects of yeast cells deleted for the same gene (Kanaar et al. 1996). This means that despite a billion years of evolutionary distance between yeast and humans, the structure and function of this gene has been at least partially conserved.

The E. coli RecA protein is the paradigmatic repair protein in recombination. S. cerevisiae has four proteins with homology to RecA and human cells at least seven. By using also the fission yeast S. pombe as a model we provided evidence for the evolutionary conservation of Rad55p, including a human homolog (Khasanov et al. 1999). This provides the structural basis to extend our regulatory studies (Rad55p phosphorylation) discussed above to mammalian cells.

4. Applied Project - EMFID - Environmental Monitoring by Fluorescence Induction and Detection

The Ames Test is still the primary method to evaluate the genotoxic potential of new compounds and to monitor for genotoxic contaminations. However, the Ames Test has severe shortfalls, because it involves a bacterial system that shows fundamental differences to eukaryotic including human cells. Moreover, the Ames Test measures a genetic endpoint which does not allow on-line measurements. Exploiting the knowledge about DNA damage checkpoints in yeast cells, we have developed a novel system that may in the future add to or possibly replace the Ames Test in genotoxic monitoring and testing.

In collaboration with Dr. Walmsley (University of Manchester Institute of Science and Technology, Manchester) we have developed a fluorescence-based system that can detect DNA damage (Walmsley et al. 1997; Billinton et al. 1998). We utilize the DNA damage-sensing capability of yeast cells that transcriptionally induce the RAD54 promoter in response to DNA damage. By replacing the RAD54 open reading frame with a variant of the green fluorescent protein, we can identify DNA damage as the cells recognizes it without waiting for a stable genetic endpoint. In addition, yeast is eukaryotic and as discussed in detail above, the basic mechanisms of cellular functions are conserved from yeasts to humans. In an EC-funded network, this principle was further developed to an on-line monitoring system for genotoxic stress. This system is currently being enhanced with regards to response time and sensitivity as well as being miniaturized and automated.