DNA helicases are key components in DNA metabolism. In the yeast Saccharomyces cerevisiae, a deletion of the Sgs1 DNA helicase causes genome instability. In human cells, mutations in orthologues of SGS1 cause Bloom, Werner, and Rothmund-Thomson syndromes that are associated with accelerated cancer and premature aging (Figure 1).
MUS81 encodes a structure-specific endonuclease. In the absence of Sgs1, Mus81 is essential since sgs1∆ mus81∆ double mutant is inviable (this phenotype is called "synthetically-lethal"). Its synthetic-lethality is suppressed by a deletion of any one of RAD52 epistasis genes, such as RAD51. That is, rad51∆ sgs1∆ mus81∆ triple mutant is viable (Table 1).
RAD51 encodes eukaryotic RecA homolog and Rad51 protein promotes homologous recombination (Figure 2).
Homologous recombination is important for DNA double-strand break repair. It is thought that the mechanism of rad51 suppression is due to removal of toxic intermediates arising during recombination. Thus, it has been thought that RAD51 is epistatic to both SGS1 and MUS81.
In the absence of RAD51, SGS1, and MUS81, cells are still alive, which means there must be other DNA repair pathway to repair DNA damage (Figure 3).
To discover new DNA repair pathway X related to these genes, we have carried out a synthetic-lethal screen using rad51∆ sgs1∆ mus81∆ triple mutant. We have identified RNH202, which encodes a subunit of RNase H2 that plays a non-essential role in Okazaki fragment processing in DNA replication.
We have been focused on the research of DNA repair network including SGS1, MUS81, RNase H2 genes, and RAD52 epistasis genes such as RAD51. Recently, it has been reported that mutation in RNase H2 causes severe neurological disease in human. Also SGS1 is responsible for genome stability and mutations in SGS1 orthologues in human cause genetic diseases that are associated with accelerated cancer and aging.
Our mission is to understand the mechanisms of maintenance of genome stability that is responsible for avoiding cancer formation and aging, and to contribute human health care through our fundamental research.
We have found that RNase H2 mutants are UV sensitive as well as rad27∆ and rad51∆ mutants, which indicates RNase H2 plays significant role in DNA repair. RNase H2 functions in parallel pathway with SGS1, MUS81, and RAD51. Its emzymatic activity is responsible for the activity in DNA repair. We also have found that RNase H2 mutants exhibit telomere elongation. We have proposed a model representing the three parallel pathways for DNA repair (Figure 4, below). Our findings shed light on the significance of RNase H2 in DNA repair and maintenance of genome stability (Ii, M. and Brill, S. J., Curr. Genet. 48, 213 (2005)).
In contrast to the general model indicating that both
SGS1 and MUS81 function downstream RAD51, our yeast genetics data have
indicated that only SGS1 functions downstream of RAD51 preferentially
and MUS81 does not.
We have proposed a new model representing the relationship between RAD51, SGS1, MUS81, and RNase H2 genes (Ii, M. and Brill, S.J., Curr. Genet. 48, 213 (2005)). We continued this project and analyzed the relationship between other RAD52 epistasis genes and SGS1, MUS81, and RNH202. We have found that there are differences between them and propose a new model to classify multiple homologous recombination pathways (Ii, M.* and Brill, S.J., manuscript in preparation. (*: corresponding author)).
Mus81 is a structure-specific endonuclease and has been sugested to function in the quality control of replication forks by preference of in-vitro substrate. In addition, the mechanism of synthetic-lethality of sgs1∆ mus81∆ double mutant is unknown. To determine the mechanism of its synthetic-lethality and in-vivo role of Mus81 in yeast, we have carried out a screen to isolate mus81 temperature-sensitive mutant in sgs1∆ background. As shown in figure 5 (with arrowheads), we have isolated a mus81 temperature sensitive mutant that cannot grow at 37˚C and analyzed the phenotypes.
We have analyzed cell cycle progression in sgs1∆ mus81ts mutant and found that cell cycle of the cells was arrested at G2/M phase at non-permissive temperature (Figure Flow Cytometory, below). We also have found that the signal transduction of DNA damage in sgs1∆ mus81ts mutant is impaired and the accumulation of damaged or stalled replication forks was suggested. Replication intermediates analysis has indicated the replication fork defects in mus81∆ cells and sgs1∆ mus81ts mutant revealed abnormal replication fork intermediates (Figure 2D gel, below). We also have found that mus81∆ mutant exhibits expansion of rDNA repeat and suggested that Mus81 functions in the maintenance of rDNA repeat expansion/contraction (Figure CHEF gel, below). Our findings determined the in-vivo role of Mus81 in quality control of replication forks and maintenance of rDNA repeat number. (Ii, M.*, Ii. T, and Brill, S.J,. Mutat. Res. 625, 1 (2007) (*:Corresponding Author)).