A yeast gene, MGS1, encoding a DNA-dependent AAA(+) ATPase is required to maintain genome stability

Abstract

Changes in DNA superhelicity during DNA replication are mediated primarily by the activities of DNA helicases and topoisomerases. If these activities are defective, the progression of the replication fork can be hindered or blocked, which can lead to double-strand breaks, elevated recombination in regions of repeated DNA, and genome instability. Hereditary diseases like Werner's and Bloom's Syndromes are caused by defects in DNA helicases, and these diseases are associated with genome instability and carcinogenesis in humans. Here we report a Saccharomyces cerevisiae gene, MGS1 (Maintenance of Genome Stability 1), which encodes a protein belonging to the AAA(+) class of ATPases, and whose central region is similar to Escherichia coli RuvB, a Holliday junction branch migration motor protein. The Mgs1 orthologues are highly conserved in prokaryotes and eukaryotes. The Mgs1 protein possesses DNA-dependent ATPase and single-strand DNA annealing activities. An mgs1 deletion mutant has an elevated rate of mitotic recombination, which causes genome instability. The mgs1 mutation is synergistic with a mutation in top3 (encoding topoisomerase III), and the double mutant exhibits severe growth defects and markedly increased genome instability. In contrast to the mgs1 mutation, a mutation in the sgs1 gene encoding a DNA helicase homologous to the Werner and Bloom helicases suppresses both the growth defect and the increased genome instability of the top3 mutant. Therefore, evolutionarily conserved Mgs1 may play a role together with RecQ family helicases and DNA topoisomerases in maintaining proper DNA topology, which is essential for genome stability.

Figure 1

Mgs1 belongs to the AAA⁺ class ATPase family, and the Mgs1 orthologues are ubiquitous in prokaryotes and eukaryotes. (A) Alignment of the conserved amino acid sequences of the members of the AAA⁺ class family (32, 33). Rfc3, S. cerevisiae Rfc3 protein (40). RuvB, E. coli RuvB protein (41). Lines above the sequences indicate the positions of the motifs characteristic of the AAA⁺ class family. (B) Alignment of amino acid sequences of the Mgs1 orthologues. Human (GenBank accession no. AF218313); S.cer, S. cerevisiae (GenBank accession no. Z71494); S.pom, Schizosaccharomyces pombe (GenBank accession no. T38421); A.tha, Arabidopsis thaliana (GenBank accession no. T00660); E.col, E. coli (GenBank accession no. P45526).

Figure 2

Overproduction of Mgs1 affects cellular sensitivity to genotoxic agents and recombination frequency. (A) Sensitivity to MMS and HU. Cells harboring empty vector (vector) or a plasmid with the galactose-inducible wild-type MGS1 gene (WT) or mutant mgs1 (K183A) gene (K183A) were grown overnight in liquid SC glucose–Leu medium to stationary phase. Cells were diluted and spotted onto SC galactose–Leu or SC glucose–Leu plates with no drug (a and b), 0.005% MMS (c and d), or 100 mM HU (e and f). The plates were incubated at 30°C for 3 days. (B) Overproduction of Mgs1 increases the intrachromosomal recombination frequency. The recombination frequencies were determined between the intrachromosomal heteroalleles in his4B-ADE2-his4X as described in Materials and Methods. The gray and black columns show the recombination frequencies of the Mgs1-repressed and the Mgs1-induced cells, respectively.

Figure 3

Effect of mgs1 mutation on growth of sgs1 and top3 strains. (A) Tetrad analysis of a genetic cross between an mgs1 strain and an mgs1 top3 strain (Lower). As a reference, tetrad analysis of a cross between a top3 strain and a TOP3 strain is shown (Upper). The genotypes of the diploids are shown on the Left. All small colonies with either mgs1 (minute) or MGS1 (small) allele were top3, which was confirmed by Ura⁺ prototropy. The spore clones were grown at 30°C for 4 days. (B) Growth curves of strains with mgs1, sgs1, or top3 mutation, or combined mutations. Yeast cells were grown to the 1 × 10⁷ cells/ml in YPAD, diluted 10-fold, and cultured at 30°C for 12 h. The cell number at each indicated time was counted under a microscope.

Figure 4

Effects of sgs1Δ and mgs1Δ mutations on the sensitivity to CPT. (A) Mgs1 overexpression increases the sensitivity of the cells to CPT. Mgs1 was overexpressed as described in the legend of Fig. 2. Cells were 10-fold serially diluted and spotted onto SC galactose–Leu plates buffered with 25 mM Hepes (pH 7.2) and containing 10 μg/ml of CPT (Right) or no CPT as a control (Left). (B) The sgs1Δ mutant is sensitive to CPT. Wild-type, sgs1Δ, and mgs1Δ cultures were 10-fold serially diluted and spotted onto YPAD plates containing 15 μg/ml CPT (Right) or no CPT as a control plates (Left).

Figure 5

DNA-dependent ATPase activity of Mgs1. (A) Purified Mgs1 proteins were analyzed by 12% SDS/PAGE and stained with Coomassie brilliant blue. (B) MgCl₂ titration of ATPase activity at 50 mM NaCl. ATPase activity of Mgs1 was assayed at different MgCl₂ concentrations in the absence of DNA (▵), in the presence of ssDNA (■) or dsDNA (●) at 30°C for 30 min. (C) NaCl titration of ATPase activity at 5 mM MgCl₂. (D) Time course of ATPase activity in the presence of 7.5 mM MgCl₂ and 25 mM NaCl. ATPase activity of wild-type Mgs1 in the absence of DNA (▵), or in the presence of M13mp18 ssDNA (■) or M13mp18 RFI (●). ATPase activity of mutant Mgs1 (K183A) in the presence of ssDNA (▴).

Figure 6

Promotion of annealing of complementary DNA strands by Mgs1. Reactions were performed as described in Material and Methods. (A) Time-course measurements of DNA annealing activity were carried out in the absence of protein (lanes 3–5), or in the presence of 150 μM Mgs1 in the absence of nucleotide cofactor (lanes 6–8) or 150 μM RecA in the presence of 2 mM ATP (lanes 9–11). Lane 1, linear pUC19 dsDNA; lane 2, heat-denatured pUC19 dsDNA. (B) Mgs1 (80 or 200 nM) was incubated with labeled heat-denatured linear pUC19 dsDNA in the absence (lanes 2–4) or presence of ATP (lanes 5–7), ATPγS (lanes 8–10), or ADP (lanes 11–13), or in the absence of Mg²⁺ (lanes 14–16) at 30°C for 15 min. The resultant samples were analyzed by electrophoresis on a 0.8% agarose gel and fluorography.