Rad62 protein functionally and physically associates with the smc5/smc6 protein complex and is required for chromosome integrity and recombination repair in fission yeast

Abstract

Smc5 and Smc6 proteins form a heterodimeric SMC (structural maintenance of chromosome) protein complex like SMC1-SMC3 cohesin and SMC2-SMC4 condensin, and they associate with non-SMC proteins Nse1 and Nse2 stably and Rad60 transiently. This multiprotein complex plays an essential role in maintaining chromosome integrity and repairing DNA double strand breaks (DSBs). This study characterizes a Schizosaccharomyces pombe mutant rad62-1, which is hypersensitive to methyl methanesulfonate (MMS) and synthetically lethal with rad2 (a feature of recombination mutants). rad62-1 is hypersensitive to UV and gamma rays, epistatic with rhp51, and defective in repair of DSBs. rad62 is essential for viability and genetically interacts with rad60, smc6, and brc1. Rad62 protein physically associates with the Smc5-6 complex. rad62-1 is synthetically lethal with mutations in the genes promoting recovery from stalled replication, such as rqh1, srs2, and mus81, and those involved in nucleotide excision repair like rad13 and rad16. These results suggest that Rad62, like Rad60, in conjunction with the Smc5-6 complex, plays an essential role in maintaining chromosome integrity and recovery from stalled replication by recombination.

FIG. 1.

Physical map of the rad62 genomic region and construction of a rad62 deletion mutant. (A) A restriction map of the rad62 region is shown at the top. The plasmid containing the rad62 deletion was constructed by replacing the HpaI-XhoI fragment in rad62 with the ura4 gene, as shown at the bottom. (B) Nucleotide sequence of the rad62 gene and predicted amino acid sequence of the Rad62 protein. The exons are underlined. Arrows show the transcription initiation sites. The poly(A) sites are shown by vertical lines. The amino acid alteration from Lys to Glu at position 223 caused by a mutation at position 1239 in rad62-1 mutant is shown.

FIG. 2.

Sequence alignment of Rad62 proteins from various organisms. Multiple-sequence alignment was performed with CLUSTAL X, version 1.81 (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/). hRad62, human Rad62 (accession number AAH27612.1); NcRad62, Neurospora crassa Rad62 (accession number E1136561.1); ScRad62, S. cerevisiae Rad62 (Qri2) (accession number CAA55925.1); CeRad62, Caenorhabditis elegans Rad62 (accession number CAB09113.1). The arrow shows the position of the rad62-1 mutation. The levels of sequence homology are shown by the heights of the blocks at the bottom.

FIG. 3.

The rad62-1 mutant is defective in DNA repair. (A and B) Sensitivities of rad62-1 mutant to UV and gamma rays. The cells at mid-logarithmic phase were irradiated with UV or gamma rays at the indicated doses, and the relative plating efficiencies were determined. Wild-type strain, MMP1 (♦); rad62-1 strain, MMP2 (▪); rhp51Δ strain, MMPX3 (▴); rad62-1 rhp51Δ strain, MMPX4 (•). The data represent the averages of the results from three experiments. (C) The rad62-1 mutant is defective in repairing DNA DSBs. Genomic DNA of wild-type and rad62-1 strains was analyzed by PFGE. Samples were taken at the indicated times (in hours) after irradiation by gamma rays at 500 Gy (+) or before irradiation (−).

FIG. 4.

Rad62 is an essential nuclear protein. (A) Tetrad analysis of rad62 heterozygous diploids. The diploid strain heterozygous for the rad62 deletion mutation was sporulated and subjected to tetrad analysis. The segregants from the dissected spores were grown on a YES plate at 30°C for 5 days. The rad62Δ segregants are boxed. (B) Terminal morphology of rad62Δ cells. The diploid strain MMPD1 was sporulated and germinated on an EMM2 plate lacking uracil. The rad62Δ cells grown for 2 days were fixed with 70% ethanol, stained with DAPI (1 μg/ml) and calcofluor white (20 μg/ml), and photographed. The abnormal nuclei are shown by arrows. (C) Nuclear localization of Rad62 protein. Cells of the rad62Δ (MMP8) carrying a plasmid for expression of GFP-Rad62 fusion protein, pGFP62, were fixed, stained with DAPI (1 μg/ml), and observed under an epifluorescence microscope. Fluorescence images of DAPI (left) and GFP-Rad62 (right) are shown.

FIG. 5.

rad62-1 is synthetically lethal with rad60-1, smc6-X and brc1Δ. rad62-1 was crossed with rad60-1, smc6-X or brc1Δ, and spores were subjected to tetrad analysis at 26°C. The double mutants are circled. Some single mutants formed small colonies, but they showed normal growth when restreaked on plates.

FIG. 6.

Suppression of MMS sensitivity of rad62-1 by overexpression of Rad60 or Brc1. rad62-1 cells (MMP7) carrying the pRAD62 plasmid, a vector plasmid, pREP41, the pRAD60 plasmid, or the pBRC1 plasmid were grown to saturation on EMM with required supplements. Serial 10-fold dilutions were spotted on YES plates with or without MMS. The plates were incubated at 30°C for 3 days.

FIG. 7.

Physical association of Rad62 with Smc5. The association of Rad62 and Smc5 in vivo was examined by coimmunoprecipitation of Rad62-FLAG-His (Rad62-FH) and Smc5-Myc. Cells expressing both tagged proteins (MMP20) or singly tagged protein (MMP21 or MMP22) were prepared and immunoprecipitated with anti-Myc or anti-FLAG. The immune complexes were separated by SDS-PAGE and immunoblotted with antibodies against Myc or FLAG. Sample volumes were adjusted to make a direct comparison of the relative amounts of Rad62-FH and Smc5-Myc in the extracts, supernatants, and precipitates possible.

FIG. 8.

Synthetic growth defect of rad62-1 with rqh1Δ, srs2Δ, mus81Δ, rad13Δ, rad16Δ, and rhp18Δ. rad62-1 was crossed with rqh1Δ, srs2Δ, mus81Δ, rad13Δ, rad16Δ, or rhp18Δ, and spores were subjected to tetrad analysis at 30°C. The double mutants are boxed. Small colonies were formed from the double mutants, rad62-1 rad13Δ and rad62-1 rhp18Δ, and they were tested for growth by streaking on plates. Some single mutants formed small colonies like the double mutants on the figure, but they showed normal growth when restreaked on plates.

FIG. 9.

Model of the Smc5-6 complex for maintenance of chromosome integrity and repair of DSBs at the replication fork. The Smc5-6 complex holds replicated chromatids in close proximity at the replication fork. Smc5 and Smc6 proteins form a heterodimeric tight complex similar to other SMC protein complexes like condensin and cohesin. Nse1, Nse2, and Rad62 associate tightly with the complex. Rad60 and Brc1 may transiently associate with the complex.