Expression of bacterial endonucleases in Saccharomyces cerevisiae mitochondria

Abstract

Expression vectors were created in which the 5′ end of the Saccharomyces cerevisiae CDC9 gene, which encodes a mitochondrial targeting peptide, was cloned in-frame with the coding regions of the EcoR I, Hind III, and Pst I endonuclease genes. Expression of the EcoR I and Hind III fusion proteins inhibited growth of yeast on glycerol-containing media and resulted in the nearly quantitative restriction digestion of their mitochondrial DNA. In contrast, expression of Pst I, which does not recognize any sites within yeast mitochondrial DNA, had no effect on growth in glycerol-containing media, and did not affect the integrity of the mitochondrial genome.

Introduction

Studies of eukaryotic DNA double-strand break (DSB) repair have been significantly advanced by the identification and characterization of mutant clones that are hypersensitive to ionizing radiation (Chu, 1997, Jackson, 2001). While exposure to ionizing radiation creates DSBs in DNA, it also results in the creation of additional types of damage including oxidative damage and DNA single strand breaks. In addition, ionizing radiation also damages other types of cellular macromolecules such as proteins and lipids (Cunniffe and O'Neill, 1999, Li et al., 2001). Investigators have therefore explored other approaches to selectively introduce DNA DSBs into eukaryotic cells (Lewis and Resnick, 2000).

One approach has involved the electroporation of purified bacterial restriction endonucleases into eukaryotic cells, most notably Chinese hamster ovary (CHO) cells. It was shown that introduction of these enzymes led to the creation of DSBs within the nuclear genome of these cells, and resulted in significant toxicity (Bryant, 1985, Giaccia et al., 1990, Costa et al., 1993, Kinashi et al., 1993). A large number of restriction endonucleases were shown to cause cell death following their electroporation into target cells. Some enzymes, such as Pvu II, are more toxic than others (Bryant et al., 1987, Kinashi et al., 1995, Harvey et al., 1997). There was no obvious correlation between a particular enzyme toxicity, and either the type of DNA DSB it introduced (i.e. blunt-end, 5′, or 3′ overhanging end), or the frequency with which the enzyme's recognition site is present in the target cell's nuclear genome (Kinashi et al., 1995). Given the limitations of transfection efficiency of proteins, there was interest in developing alternate ways to express bacterial endonucleases in mammalian cells.

A variety of groups utilized molecular cloning and gene transfer techniques to accomplish this objective. Expression of the gene encoding the yeast endonuclease I-Sce I in mammalian, plant, and yeast cells resulted in site-specific DNA DSBs (Plessis et al., 1994, Puchta et al., 1993, Lukacsovich et al., 1994, Rouet et al., 1994, Segal and Carroll, 1995, Brenneman et al., 1996, Anglana and Bacchetti, 1999, Richardson et al., 1999). Transfer and expression of the genes encoding endonucleases I-PpoI and I-Cre I, derived from Physarum polycephalum and Chlamydomonas reinhardtii, respectively, in human cells created site-specific DSBs in nuclear DNA (Monnat et al., 1999). The bacterial endonuclease EcoR I gene has been expressed in yeast cells and shown to create nuclear DNA DSBs (Barnes and Rine, 1985, Barnes and Rio, 1997, Lewis et al., 1998, Lewis et al., 1999).

The repair of restriction endonuclease-induced DNA DSBs has been examined in mutant and wild-type (WT) cells. In yeast, for example, cells deficient in Rad52p, a mutation previously shown to render yeast 100-fold more sensitive to ionizing radiation, were unable to grow following nuclear expression of restriction endonuclease EcoR I (Resnick, 1969, Barnes and Rine, 1985). Additionally, it was determined that the Ku heterodimer, a protein complex later shown to be essential in non-homologous DNA end-joining, was necessary for the repair of nuclear DNA DSBs introduced by expression of the gene encoding EcoR I (Milne et al., 1996, Barnes and Rio, 1997). It was shown that similar expression of EcoR I was lethal in yeast cells devoid of the Rad50, Mre11, and Xrs2 proteins, which have been shown to be involved in non-homologous DNA end-joining (Lewis et al., 1999). Thus, it has been demonstrated that non-homologous DNA end-joining is an indispensable mechanism for the specific repair nuclear DSBs in yeast. Similarly, CHO cells deficient in the 86 kDa subunit of the hamster Ku heterodimer were hypersensitive to the cytotoxic effects of endonucleases electroporated into them, compared to WT cells. Liang et al. observed that chromosomal end-joining of a DSB induced by intracellular expression of I-Sce I was impaired in Ku-deficient hamster cells (Liang et al., 1996).

To examine the consequences of induced DSBs within mitochondrial DNA, targeted expression of restriction endonucleases to this organelle would be a valuable tool. It has been demonstrated that the EcoR I endonuclease could be directed to the mitochondria of Saccharomyces cerevisiae (Donahue et al., 2001). Here we show that heterlogous fusion genes are created by placing the mitochondrial targeting sequence (MTS) from the CDC9 DNA ligase gene upstream and in-frame with three endonucleases, EcoR I, Hind III, and Pst I. Extracts made from yeast expressing the targeted endonucleases show that all three endonucleases are functional. We demonstrate that expression of targeted endonucleases that have recognition sites within the mitochondrial genome (EcoR I and Hind III) causes a growth arrest phenotype in yeast grown in glycerol-containing media. Expression of Pst I, which has no recognition sites in the mitochondrial genome, does not result in this growth arrest. Finally, we confirm that DSBs are created in mitochondrial DNA in vivo in yeast expressing both EcoR I and Hind III, while mitochondrial DNA remains intact in yeast expressing Pst I.