Regulation of error-prone translesion synthesis by Spartan/C1orf124

Abstract

Translesion synthesis (TLS) employs low fidelity polymerases to replicate past damaged DNA in a potentially error-prone process. Regulatory mechanisms that prevent TLS-associated mutagenesis are unknown; however, our recent studies suggest that the PCNA-binding protein Spartan plays a role in suppression of damage-induced mutagenesis. Here, we show that Spartan negatively regulates error-prone TLS that is dependent on POLD3, the accessory subunit of the replicative DNA polymerase Pol δ. We demonstrate that the putative zinc metalloprotease domain SprT in Spartan directly interacts with POLD3 and contributes to suppression of damage-induced mutagenesis. Depletion of Spartan induces complex formation of POLD3 with Rev1 and the error-prone TLS polymerase Pol ζ, and elevates mutagenesis that relies on POLD3, Rev1 and Pol ζ. These results suggest that Spartan negatively regulates POLD3 function in Rev1/Pol ζ-dependent TLS, revealing a previously unrecognized regulatory step in error-prone TLS.

Figure 1.

The SprT domain of Spartan is a putative zinc metalloprotease and Glu112 is required for Spartan to suppress UV-induced mutagenesis. (A) Schematic representation of the domain structure of human Spartan. PIP, PCNA-interacting peptide; UBZ4, ubiquitin-binding zinc-finger 4. (B) Alignment of the SprT domain sequences from Spartan homologs in selected species. The HExxH motif is indicated above the sequences. (C) Model of a truncated human SprT domain (residues 43–121; generated by the Phyre server) showing the three residues of the HExxH motif (left); crystal structure of MMP8 (Protein Data Bank ID: 2OY4) showing the three residues of the HExxH motif and the zinc divalent cation coordinating three histidines (middle); overlay of the truncated SprT and MMP8 revealing the structural similarity in the active site between the two proteins (right). (D) Glu112 is required for suppression of UV-induced mutagenesis by Spartan. UV-induced mutagenesis was measured using the SupF shuttle vector system in 293 T cells transfected with the indicated siRNA oligos. Mutation frequencies are presented as percentage of mutant SupF genes. Error bars represent SD (n = 3). Wild-type or the E112A mutant of Spartan was expressed with 3xFlag tag in an siRNA-resistant form. Spartan proteins were analysed by Western blotting. Positions of endogenous and exogenous Spartan proteins are indicated by white and black arrowheads, respectively.

Figure 2.

The SprT domain of Spartan interacts with Pol δ subunit POLD3. (A) The E112A mutant of SprT captures Pol δ in vivo. Wild-type or the E112A mutant of SprT proteins were stably expressed in 293A and purified on anti-Flag beads. Copurified endogenous proteins were analysed by Western blotting for the indicated Pol δ subunits. (B) The SprT domain captures co-expressed POLD3 in vivo. Wild-type or the E112A mutant of SprT proteins were transiently expressed in 293T with each Pol δ subunit tagged with 4HA and immunoprecipitated with anti-Flag beads. Precipitated proteins were analysed by Western blotting for the indicated proteins. (C) Interactions between full-length Spartan and POLD3. Experiments were performed as in (B) except that full-length Spartan was expressed. (D) Direct interaction of the Spartan SprT domain with POLD3. In vitro GST-pull down assays were performed using the indicated recombinant proteins.

Figure 3.

POLD3 is involved in error-prone TLS in Spartan-depleted cells. (A) POLD3 is required for mutagenesis in Spartan-depleted cells. Cells were treated as in Figure 1D. Error bars represent SD (n = 3). Knockdown of Spartan and POLD3 was confirmed by Western blotting. (B) Mutagenesis in Spartan-depleted cells is dependent on Rev1 and Pol ζ. Error bars represent SD (n = 3). Knockdown of Spartan and Rev1 was confirmed by Western blotting. #Cross-reactive bands. Action of Rev3 siRNA was confirmed in Figure S4.

Figure 4.

Spartan depletion induces complex formation of POLD3 with Rev1 and Pol ζ. (A) Spartan depletion induces interaction of POLD3 with Rev1. Cells stably expressing 3xFlag-POLD3 at near physiological levels were transfected with the indicated siRNAs. 3xFlag-POLD3 proteins were immunoprecipitated and Western blotting was performed for the indicated proteins. RNAi of Rev1 shows the specificity of anti-Rev1 antibodies. #Cross-reactive bands. (B) Spartan depletion induces interaction of Rev1 and POLD3. Experiments were performed similarly in cells stably expressing 3xFlag-Rev1 after transfection with the indicated siRNAs. (C) Spartan depletion induces interaction of POLD3 with Rev7. Experiments were performed as in (A) and Western blotting was performed for the indicated proteins.