SUMO modification of Rad22, the Schizosaccharomyces pombe homologue of the recombination protein Rad52

Abstract

The Schizosaccharomyces pombe rad31 and hus5 genes are required for the DNA damage response, as mutants defective in these genes are sensitive to DNA damaging agents, such as UV and ionising radiation and to the DNA synthesis inhibitor hydroxyurea (HU). Sequence analysis has suggested that rad31 and hus5 encode components of the Pmt3 (SUMO) modification process in S.pombe. We show here that the rad31 null and hus5.62 mutants display reduced levels of Pmt3 modification. We have initiated a search for proteins required for the DNA damage response, which may be modified by Pmt3 and have identified Rad22, the fission yeast homologue of the recombination protein Rad52. Purification of myc + His-tagged Rad22 protein from cells expressing HA-tagged Pmt3 identifies an 83 kDa species which cross-reacts with anti-HA antisera. We show here that Rad22 interacts with Rhp51 and Rpa70 (the fission yeast homologues of Rad51 and the large subunit of RPA, respectively), but that neither of these proteins appears to be responsible for the 83 kDa species. The 83 kDa species is observed when extracts are prepared under both native and denaturing conditions, and is also observed when myc + His-tagged Rad22 and Pmt3 are expressed at wild type levels, suggesting that Rad22 is modified by Pmt3 in vivo. We have established an S.pombe in vitro Pmt3 modification system and have shown that Rad22 and Rhp51 are modified in vitro, but that Rpa70 is not.

Figure 1

Pmt3 modification in S.pombe. (A) Schematic showing the components of the S.cerevisiae and S.pombe Pmt3 modification pathway. The S.cerevisiae nomenclature is taken from previous studies (5,16). (B) Anti-Pmt3 antisera are specific for Pmt3 and do not recognise ubiquitin. NΔPmt3 (lanes 1 and 3) and ubiquitin (0.5 µg) (lanes 2 and 4) were separated by SDS–PAGE (12.5%) and western blotted with anti-Pmt3 antisera (lanes 1 and 2) or anti-ubiquitin antisera (lanes 3 and 4). (C) SDS–PAGE and western blotting with anti-Pmt3 antisera of protein extracts from wild type (sp.011) lane 1, rad31.d (sp.333) lane 2 and hus5.62 (sp.376) lane 3.

Figure 2

Interaction of Rad22 with Pmt3, Rhp51 and Rpa70. (A) Interaction with Pmt3. (B) Interaction with Rhp51. (C) Interaction with Rpa70. In all cases wild type cells (sp.011) were transformed with plasmids expressing the indicated proteins as tagged fusion proteins under the control of the nmt1 promoter. Protein interactions were investigated using Ni²⁺ agarose beads, TCA extracts were used as controls to check expression levels. Samples were analysed by SDS–PAGE and western blotted with anti-myc or anti-HA antisera as indicated. *, New HA-cross-reacting species.

Figure 3

Interaction of Rad22 with Hus5 and Pmt3. (A) Bacterial cell lysates were mixed as indicated and proteins were fractionated on glutathione–Sepharose. Proteins binding to glutathione (GST-pull down), crude extract and unbound material (supernatant) were separated by SDS–PAGE and western blotted with anti-His antisera or stained with Coomassie Brilliant Blue. (B) Interaction of Rad22 and Pmt3 was analysed using extracts from cells over-expressing both proteins as in Figure 2A, but in the presence of 6 M guanidine–HCl. (C) Interaction of Rad22 and Pmt3 expressed at wild type levels. Strains used were an integrated myc + His-tagged Rad22 strain (sp.568) and sp.011 as the negative control. Samples were analysed by SDS–PAGE and western blotted with anti-myc or anti-Pmt3 antisera as indicated. Lanes 1–4, 7 and 8 were exposed to X-ray film for 10 s, while lanes 5 and 6 were exposed for 30 min.

Figure 4

Schizosaccharomyces pombe in vitro Pmt3 modification system. (A) Protein components used to establish the assay. Coomassie stain of SDS–PAGE. Lane 1, 4 µg Hus5; lane 2, 10 µg His-Pmt3-GG; lane 3, 1 µg GST-Rad31, GST-Fub2. (B) Modification of PML. Assays were set up as described in the Materials and Methods; all samples were incubated in assay buffer for 2 h at 30°C. Lanes 1–7, wild type PML; lanes 8 and 9, PML-3K-R. Lanes 1 and 2, all assay components; lane 3, no Hus5; lane 4, no Pmt3; lane 5, no Rad31 + Fub2; lane 6, no added ATP; lanes 7 and 9, no Hus5, Pmt3, Rad31 and Fub2 (C–E) Testing for modification of Rad22 (C), Rhp51 (D) and Rpa70 (E). Addition of components was as indicated. (C) *, Major Pmt3-modified product; <, minor Pmt3-modified species of 105 kDa.