The modification of proliferating cell nuclear antigen (PCNA) by small ubiquitin-like modifier (SUMO) recruits Srs2 during S phase (Hoege et al., 2002; Pfander et al., 2005). The recruitment of Srs2 to replication fork through physical interaction between the C-terminus of Srs2 (residues 1038–1174) and SUMOylated PCNA regulates homologous recombination by disrupting the Rad51 nucleoprotein filament formation, which is an essential step in a recombination pathway (Krejci et al., 2003; Veaute et al., 2003; Pfander et al., 2005; Colavito et al., 2009). Although many studies stressed the importance of the interaction between Srs2 and SUMOylated PCNA in the regulation of homologous recombination, the mechanism of the recognition of SUMOylated PCNA by Srs2 remains unclear at the molecular level.
To elucidate the mechanism of recognition of SUMOylated PCNA by Srs2, we first hypothesized that Srs2 might contain a PCNA-interacting motif nearby the SUMO-binding motif (SBM; residues 1170–1173; Kerscher, 2007) to distinguish the SUMOylated PCNA from other SUMOylated substrates in the nucleus. To check the existence of a PCNA-interacting motif, we performed pull-down assays using TrxHisSrs2/137c (residues 1038–1174; known as the SUMOylated PCNA-binding region; Pfander et al., 2005), TrxHisSrs2/137cΔC32 (residues 1038–1142) and TrxHisSrs2/32c (residues 1143–1174), which contain thioredoxin (Trx) and hexahistidine (His) tags at the N-terminus. PCNA co-eluted with TrxHisSrs2/137c and TrxHisSrs2/32c, not with TrxHisSrs2/137cΔC32 (Figure 1A). We further acquired sequential heteronuclear single quantum coherence (HSQC) spectra of 15N-labeled Srs2/32c with various concentrations of unlabeled PCNA. The peaks of Srs2/32c disappeared when unlabeled PCNA was added, and the most perturbed region of Srs2/32c was located N-terminal to the SBM, within residues 1149–1162 (Supplementary Figure S1).