Insights into the regulation of human Rev1 for translesion synthesis polymerases revealed by the structural studies on its polymerase-interacting domain.

Dear Editor, Translesion synthesis (TLS) allows the DNA replication machinery to bypass an unrepaired DNA damage site using special polymerases called TLS polymerases (Fischhaber and Friedberg, 2005). When compared with the replicative polymerases, TLS polymerases have comparatively large active sites to incorporate the base opposite the damaged DNA and low fidelity to ensure progression of synthesis using the damaged template (McCulloch and Kunkel, 2008). Though TLS rescues the cells from the collapse of the replication fork, the bypass of the lesions can be a potential cause for the mutation generation (Wang, 2001). Therefore, tight regulation of the TLS polymerases is extremely important. Recent research shows that Rev1 can act as a regulator and defines distinct mechanism for TLS when compared with PCNA (Edmunds et al., 2008; Hendel et al., 2011). In comparison to other TLS polymerases, the catalytic function of Rev1 is not required for the mutagenic DNA damage tolerance (Lawrence, 2004; Prakash et al., 2005). Instead the C-terminus of Rev1, which interacts with TLS polymerases k, h, i and z (consisting of Rev3 and Rev7) in eukaryotes (Murakumo et al., 2001; Guo et al., 2003; Ohashi et al., 2004), is reported to be required for the DNA damage tolerance (D’Souza et al., 2008). Therefore, the understanding of the molecular basis of the C-terminus of Rev1 and its related interactions with TLS polymerases is important in comprehending the mechanism of the TLS polymerases regulation. Here, we report the first structural studies on the TLS polymerase-interacting domain of human Rev1 [hRev1-polymerase-interacting domain (PID)] and its interactions with TLS polymerases k, h, i and z. In order to understand how Rev1 recruits the TLS polymerases, the complex structure of hRev1-PID with Pol k 562–577 was first characterized using NMR spectroscopy (Supplementary Table S1). The overall structure of bound hRev1-PID consists of four a-helices oriented anti-parallel to each other and formed a four a-helix bundle. The two turned a-helix of Pol k formed by residues from F567 to E574 contacts the surface formed by a1, a2 and the rigid N-terminus of hRev1-PID (Figure 1A). Further double titration experiments among the polymerase k, h and i showed that at same concentration Pol k can completely replace the Pol h and i for the Rev1 interaction (Supplementary Figure S1A and B), therefore, the competition binding observed here indicates a shared binding interface on Rev1 for Pol k, h and i. In addition, similar heteronuclear single quantum coherence patterns (Supplementary Figure S1C) and dynamic properties (Supplementary Table S2) have been observed for 15N-hRev1-PID bound with polymerase k, h and i, suggesting that hRev1-PID, when interacting with Pol h and Pol i adopts a structure similar to when it binds with Pol k. To further elucidate the recruitment manner of Rev1 binding to Pol z, the complex structure between hRev1-PID and Pol z (Rev7 – and Rev3 – ) was solved using crystallography at 1.9 Å resolution (Supplementary Table S3). The hRev1-PID in complex with Pol z remains the identical conformation compared with its bound form with Pol k with R.M.S. deviation of 0.7 Å (Supplementary Figure S2A and B). Based on our complex structure, the binding interface for Pol z locates in the N-terminal end of a3 and C-terminal end of a4 on hRev1-PID (Figure 1B), which is different from the binding sites for Pol k. Taken together, Rev1 uses the same tertiary structure and two surfaces to recruit TLS polymerases, with one site for polymerase k, h, i and the other site for polymerase z. To shed light on the recognition mechanism between Rev1 and TLS polymerases, the Rev1-interacting motifs (RIM) of polymerase k, h, i and z were investigated. Previous report showed that two highly conserved FF-motif is important for the Rev1 recognition (Ohashi et al., 2009). However, based on the sequence alignment on the Rev1-interacting region of polymerase k, h and i, we found that K571 is also highly conserved (Figure 1C). Further mutation studies on the Pol k K571G or hRev1-PID E1174K showed the significant decrease of binding affinity to hRev1-PID and Pol k, respectively, indicating that K571 is indispensable for the Rev1 recognition through the electrostatic interaction with E1174 of Rev1 (Figure 1D). Therefore, we refer the ‘xxxFFxxK’ (where x represents any residue) as the RIM of Pol k, h, i. On the basis of our complex structure, F568 of the Pol k fits into the deep hydrophobic pocket formed by L1159, L1171, L1172, W1175 and V1190 of Rev1, while F567 of the Pol k is involved in the hydrophobic interaction with W1175 of Rev1 and the side chain of K571 of Pol k (Supplementary Figure S3). Further electrostatic surface analysis reveals that the RIM of polymerases k, h and i is recognized by Rev1 through the unmarked hydrophobic pocket acting as the ‘lock’ 204 | Journal of Molecular Cell Biology (2013), 5, 204–206 doi:10.1093/jmcb/mjs061 Published online December 6, 2012