Miki Ii Lab -Research-

INTRODUCTION

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.

PREVIOUS STUDIES

1. Role of RNase H2 in DNA repair

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)).

2. Classification of multiple homologous recombination pathways that are regulated by

downstream DNA repair pathways

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)).

3. in-vivo role of Mus81 in the maintenance of replication forks at rDNA and the rDNA

repeat number

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)).

RESEARCH PLAN

1. DNA repair and genome stability controlled by protein SUMOylation.

Target proteins: related to cancer and aging.

Recently, it has been reported that many proteins of DNA repair and genome stability are controlled by SUMO (small ubiquitin-like modifier) modification in response to cell cycle progression and DNA damage in eukaryotes. For example, SUMO-modified PCNA was found to interact directly and preferentially with Srs2 helicase to prevent recombination during S-phase in yeast (B. Pfander et al., Nature 436, 428 (2005)), in contrast to the functions of unmodified, mono-ubiquitylated- or poly-ubiquitylated PCNA. SUMO is related to ubiquitin and conjugated to various proteins to change the properties of modified proteins. In yeast, SUMO precursor is processed and activated by E1 enzyme (Aos1 and Uba2). E2 SUMO conjugating enzymes (Ubc9) ligates SUMO to the target protein with the help of E3 ligase.

SUMO is also deconjugated by SUMO-specific proteases and the history of SUMO conjugation will remain even after deconjugated, to control the fate of the proteins. It is necessary to determine what proteins are SUMOylated and what feature of the proteins is changed by SUMO modification for understanding the mechanisms of DNA repair and the maintenance of genome stability in-vivo.
We will target the proteins that are related to cancer and aging. By studying in-vivo SUMOylation of those proteins that are responsible for avoiding cancer and accelerated aging, we will be involved in "Biomedical Research" and our aim is to contribute human health care through our study on protein SUMOylation and ubiquitylation.

2. Classification of multiple homologous recombination pathways.

We have been studying homologous recombination pathways and related DNA repair pathways by using yeast genetics. In our previous work, 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)). Recently, we have further analyzed the relationship between other epistasis genes and RAD52, SGS1, MUS81, and RNH202 and updated our model (Ii, M.* and Brill, S.J., manuscript in preparation (*: corresponding author)). We will continue this project and aim to find out the network of homologous recombination by applying molecular biological and biochemical approaches. Our unique proposal in this field will determine the in-vivo feature of homologous recombination and related DNA repair pathways.

3. Role of RNase H2 in DNA repair and maintenance of genome stability.

    We have reported that RNase H2 plays significant role in DNA repair, especially in the repair of DNA damage caused by UV irradiation (Ii, M. and Brill, S.J., Curr. Genet. 48, 213 (2005)). Also we have found that RNase H2 functions in a parallel pathway with RAD51, SGS1, and MUS81 in DNA repair. RNase H2 is conserved through evolution. The role of RNase H2 has been thought to help the removal of Okazaki fragment primers in DNA replication, by coordination with Rad27 that is the major enzyme functioning in removal of Okazaki fragment primers. Without external DNA damage such as UV irradiation, yeast RNase H2 mutants are healthy and that is why RNase H2 is thought not playing important role in eukaryotic cells.
    Recently, it has been reported that a mutation in human RNase H2 genes cause neurological disease Aicardi-Goutieres syndrome (AGS) (Crow, Y.J. et al. Nat. Genet., (2006)). This finding has indicated that the significance of RNase H2 in human cells.
    To determine in-vivo function of RNase H2 in eukaryotes, we will push on this project by applying molecular biological and biochemical approaches.

- Our research -

In our lab we will be focused on the projects as described above. As drawn below, we will define "the DNA repair network" that is responsible for avoiding cancer formation and accelerated aging.
By adding protein modification such as SUMOylation and ubiquitylation, we will determine in-vivo feature of DNA repair and maintenance of genome stability in detail and be involved in "Biomedical Research".

Department of Biological Sciences, University of Alaska Anchorage

Miki Ii Lab