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Comparative Study
. 2005 Jun 1;24(11):2024-33.
doi: 10.1038/sj.emboj.7600684. Epub 2005 May 12.

RMI1/NCE4, a suppressor of genome instability, encodes a member of the RecQ helicase/Topo III complex

Michael Chang  1 , Mohammed Bellaoui, Chaoying Zhang, Ridhdhi Desai, Pavel Morozov, Lissette Delgado-Cruzata, Rodney Rothstein, Greg A Freyer, Charles Boone, Grant W Brown
Affiliations
Comparative Study

RMI1/NCE4, a suppressor of genome instability, encodes a member of the RecQ helicase/Topo III complex

Michael Chang et al. EMBO J. .

Abstract

SGS1 encodes a DNA helicase whose homologues in human cells include the BLM, WRN, and RECQ4 genes, mutations in which lead to cancer-predisposition syndromes. Clustering of synthetic genetic interactions identified by large-scale genetic network analysis revealed that the genetic interaction profile of the gene RMI1 (RecQ-mediated genome instability, also known as NCE4 and YPL024W) was highly similar to that of SGS1 and TOP3, suggesting a functional relationship between Rmi1 and the Sgs1/Top3 complex. We show that Rmi1 physically interacts with Sgs1 and Top3 and is a third member of this complex. Cells lacking RMI1 activate the Rad53 checkpoint kinase, undergo a mitotic delay, and display increased relocalization of the recombination repair protein Rad52, indicating the presence of spontaneous DNA damage. Consistent with a role for RMI1 in maintaining genome integrity, rmi1Delta cells exhibit increased recombination frequency and increased frequency of gross chromosomal rearrangements. In addition, rmi1Delta strains fail to fully activate Rad53 upon exposure to DNA-damaging agents, suggesting that Rmi1 is also an important part of the Rad53-dependent DNA damage response.

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Figures

Figure 1
Figure 1
rmi1Δ mutants exhibit a growth defect that can be suppressed by mutation of SGS1. (A) The rmi1Δ∷kanMX6 strain was backcrossed to a wild-type strain (BY4741). The resulting diploids were sporulated and tetrads were dissected on YPD. Each column represents the four spores from a single tetrad. The genotypes of the resulting colonies are indicated with circles (○) for rmi1Δ∷kanMX6. (B) SGAM analysis using an rmi1Δ∷natMX6 query strain (which contains supX) revealed a set of colinear synthetic genetic interactions on chromosome XIII. A red bar indicates that deletion of the corresponding gene resulted in a genetic interaction. Black bars represent essential genes, which are not a part of the gene deletion collection. Gray bars indicate ORFs for which no deletion mutant was made as part of the Saccharomyces Gene Deletion Project (Winzeler et al, 1999) and genes that are often found in control screens using a wild-type query strain, and therefore are filtered from the results of SGA analyses. (C) An rmi1Δ∷natMX6 strain lacking supX was crossed to an sgs1Δ∷kanMX6 strain. The resulting diploids were sporulated for tetrad analysis as in panel A. The genotypes of the resulting colonies are indicated with boxes (□) for sgs1Δ∷kanMX6 and circles (○) for rmi1Δ∷natMX6.
Figure 2
Figure 2
Rmi1 physically associates with the Sgs1/Top3 complex. (A, B) Extracts from yeast strains expressing the indicated epitope-tagged proteins were immunoprecipitated with IgG agarose. In all, 10% of the input extract (E) and the immunoprecipitate (IP) was fractionated by SDS–PAGE. Immunoblots were probed with anti-HA antibody to detect Sgs1, with anti-VSV antibody to detect Top3, or with peroxidase–anti-peroxidase to detect Rmi1-TAP. (C) Extract from a yeast strain expressing Sgs1-HA, Top3-VSV, and Rmi1-TAP was fractionated on a Superose 6 gel filtration column. Fractions were precipitated with TCA and analyzed by immunoblotting. The elution positions of molecular weight standards are indicated, as is the void volume of the column (Vo). (D) Extracts from yeast strains expressing Sgs1-HA and Top3-TAP or Sgs1-HA and Rmi1-TAP in an rmi1Δ or top3Δ background, respectively, were immunoprecipitated with IgG agarose to precipitate the TAP-tagged protein (lanes marked T) or with unconjugated agarose as a control (lanes marked C). The precipitates were immunoblotted and probed with anti-HA antibodies to detect Sgs1-HA (top panel) or with peroxidase–anti-peroxidase to detect the TAP-tagged proteins. (E) sgs1Δ, sgs1Δ rmi1Δ, and sgs1Δ top3Δ strains were transformed with empty vector (vector) or low-copy plasmids expressing HA-tagged Sgs1 (Sgs1) or helicase-dead Sgs1 (Sgs1-hd). TCA-fixed extracts were prepared and fractionated by SGS–PAGE. Immunoblots were probed with anti-HA antibody to detect Sgs1 or Sgs1-hd, and with anti-tubulin antibodies as a loading control.
Figure 3
Figure 3
rmi1Δ mutants exhibit Rad53 checkpoint activation during an unperturbed cell cycle. (A) Asynchronous cultures of wild type (WT), rmi1Δ, top3Δ, sgs1Δ, rmi1Δ sgs1Δ, and top3Δ sgs1Δ were examined microscopically to determine the % of cells with a bud. (B) Logarithmically growing cultures were arrested in G1 with alpha factor and released into fresh YPD media. At the indicated times, samples were fixed with TCA, extracts were fractionated on SDS–PAGE, and immunoblotted to detect Rad53. The position of the activated phosphorylated Rad53 is indicated. (C) Samples prepared as in panel B were fractionated on SDS–PAGE for in situ kinase assay of Rad53 (upper panel). A parallel blot was probed with anti-tubulin antibody as a loading control (lower panel). (D) An rmi1Δ∷kanMX6 strain was crossed to a rad53-11∷URA3 strain. The resulting diploids were sporulated and tetrads were dissected on YPD. The genotypes of the resulting colonies are indicated with boxes (□) for rmi1Δ∷kanMX6 and with circles (○) for rad53-11∷URA3. Inferred double mutants are indicated with a box and circle.
Figure 4
Figure 4
Spontaneous Rad52 focus formation in rmi1Δ cells. (A) Logarithmically growing cells expressing Rad52-YFP were visualized by fluorescence microscopy. For each pair of images, the left panel is a DIC image and the right panel is a fluorescence image showing Rad52-YFP. Representative cells are shown. (B) The percentage of cells with Rad52 foci was determined for the indicated strains. G1 cells with Rad52 foci are represented by the gold bars and S/G2/M cells with Rad52 foci are represented by the blue bars. WT, wild type.
Figure 5
Figure 5
Deletion of RMI1 causes genomic instability. (A) Recombination rate was measured using a direct repeat recombination assay. The average and standard deviation of three fluctuation tests are shown for each strain. (B) GCR rate was measured. The average and standard deviation of four fluctuation tests are shown for each strain. WT, wild type.
Figure 6
Figure 6
Rmi1 is required for the DNA damage response. (A) Serial dilutions (10-fold) of cultures of the indicated mutants were spotted on YPD, YPD containing 0.004% (v/v) MMS, or YPD containing 10 mM HU. All plates were incubated at 30°C for 2–3 days. (B) Logarithmically growing cultures of the indicated mutants were incubated in YPD containing 0.004% (v/v) MMS or 10 mM HU at 30°C. At the indicated times, samples were withdrawn and plated on YPD to determine the number of viable cells. The percentage of viable cells relative to the number of viable cells at t=0 is shown. Plots represent the average of three experiments, and error bars span 1 s.d. (C) Logarithmically growing cultures were arrested in G1 with alpha factor and released into medium containing either 0.035% (v/v) MMS or 150 mM HU. At the indicated times, samples were fixed and extracts fractionated by SDS–PAGE. Following transfer, the immunoblot was probed with anti-Rad53 antibody. WT, wild type.
Figure 7
Figure 7
Rmi1 homologues. (A) Schematic diagrams of Rmi1 homologues from S. cerevisiae (Sc), S. pombe (Sp), Homo sapiens (Hs), and Mus musculus (Mm). Regions of high sequence identity are indicated by the three shaded boxes. (B) S. pombe rmi1+ is a functional homologue of RMI1. rmi1Δ∷G418R rqh1Δ∷ura4+ was crossed to rmi1+rqh1+ and tetrads were dissected on YE5S. The genotypes of the resulting colonies are indicated with boxes (□) for (inferred) rmi1Δ∷G418R and with circles (○) for rqh1Δ∷ura4+. (C) Micrographs of rmi1Δ∷G418R rqh1Δ∷ura4+ microcolonies from panel C.
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References

    1. Ajima J, Umezu K, Maki H (2002) Elevated incidence of loss of heterozygosity (LOH) in an sgs1 mutant of Saccharomyces cerevisiae: roles of yeast RecQ helicase in suppression of aneuploidy, interchromosomal rearrangement, and the simultaneous incidence of both events during mitotic growth. Mutat Res 504: 157–172 - PubMed
    1. Bellaoui M, Chang M, Ou J, Xu H, Boone C, Brown GW (2003) Elg1 forms an alternative RFC complex important for DNA replication and genome integrity. EMBO J 22: 4304–4313 - PMC - PubMed
    1. Bennett RJ, Noirot-Gros MF, Wang JC (2000) Interaction between yeast sgs1 helicase and DNA topoisomerase III. J Biol Chem 275: 26898–26905 - PubMed
    1. Bjergbaek L, Cobb JA, Tsai-Pflugfelder M, Gasser SM (2005) Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance. EMBO J 24: 405–417 - PMC - PubMed
    1. Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P, Boeke JD (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14: 115–132 - PubMed

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