Cell-cycle-dependent localisation of Ulp1

In S. pombe, processes required for the maintenance of genetic integrity have been defined through the analysis of radiationsensitive mutants and the subsequent identification of genes involved in a range of different DNA damage responses, for example, nucleotide excision repair, recombination repair and DNA integrity checkpoints (Carr et al., 1994; Caspari and Carr, 1999; Murray et al., 1992; Tavassoli et al., 1995). During our characterisation of these processes we identified the rad31.1 mutant (Shayeghi et al., 1997), which we demonstrated to be epistatic with hus5.62 (al-Khodairy et al., 1995). These two mutants exhibit a moderate sensitivity to DNA damaging agents such as UV and ionising radiation and to the DNA synthesis inhibitor, hydroxyurea (HU) (al-Khodairy et al., 1995; Shayeghi et al., 1997). The mutants also display similar defects in cell and nuclear morphology, displaying a frequent ‘cut’ phenotype in the absence of DNA damage and the occurrence of chromosome fragmentation. Cells display a slow growth phenotype and increased mini-chromosome loss compared with wild-type cells. Null alleles of rad31 and hus5 have similar, but somewhat more severe, phenotypes. The S. pombe rad31 and hus5 genes, which were initially proposed to be involved in SUMO modification as a result of their high level of sequence identity to the S. cerevisiae AOS1 and UBC9 genes (al-Khodairy et al., 1995; Johnson et al., 1997; Shayeghi et al., 1997; Tanaka et al., 1999), have recently been shown to be required for SUMO modification by their activity in an in vitro SUMO (Pmt3) modification system (Ho et al., 2001). Specifically, the rad31 and hus5 genes encode one subunit of the heterodimeric activator and the conjugator, respectively, for the S. pombe SUMO protein, Pmt3. Pmt3 is a member of a Ubl (ubiquitin-like) family, which has been identified independently in many organisms, and its members are known variously as SUMO, PIC1, sentrin and UBL1 in mammalian cells, as Smt3 in S. cerevisiae and as Pmt3 in S. pombe (Yeh et al., 2000). The Ubls are expressed as precursor molecules that, like ubiquitin, require C-terminal processing to reveal the double glycine (GG) motif needed for conjugation to the target lysine residue (Johnson et al., 1997). The conjugation of SUMO and other Ubls such as Rub1/NEDD8 to substrate proteins is similar to that of ubiquitination and requires activation and conjugation of the Ubl by E1 and E2 enzymes (Desterro et al., 1997; Johnson and Blobel, 1997). Conjugation is initiated by an activation enzyme (E1), which, in an ATP-dependent reaction, forms a thiolester linkage with the Ubl. The Ubl is then transferred to a conjugating enzyme (E2), forming a similar thiolester linkage, before transfer to a target lysine residue of the substrate protein. Unlike the E1 required for ubiquitin activation, which is a monomer, the E1 (or SAE) required for SUMO activation is a heterodimer in which the two subunits are related to the Nand C-terminal domains of the ubiquitin E1 enzymes (Johnson et al., 1997). In S. pombe, the Rad31 protein acts in conjunction with Fub2 (Tanaka et al., 1999) to form the E1 heterodimer (Tanaka et al., 1999; Ho et al., 2001). 1113