Eukaryotic DNA polymerases require an iron-sulfur cluster for the formation of active complexes

Abstract

The eukaryotic replicative DNA polymerases (Pol α, δ and ɛ) and the major DNA mutagenesis enzyme Pol ζ contain two conserved cysteine-rich metal-binding motifs (CysA and CysB) in the C-terminal domain (CTD) of their catalytic subunits. Here we demonstrate by in vivo and in vitro approaches the presence of an essential [4Fe-4S] cluster in the CysB motif of all four yeast B-family DNA polymerases. Loss of the [4Fe-4S] cofactor by cysteine ligand mutagenesis in Pol3 destabilized the CTD and abrogated interaction with the Pol31 and Pol32 subunits. Reciprocally, overexpression of accessory subunits increased the amount of the CTD-bound Fe-S cluster. This implies an important physiological role of the Fe-S cluster in polymerase complex stabilization. Further, we demonstrate that the Zn-binding CysA motif is required for PCNA-mediated Pol δ processivity. Together, our findings show that the function of eukaryotic replicative DNA polymerases crucially depends on different metallocenters for accessory subunit recruitment and replisome stability.

Figure 1. Yeast replicative DNA polymerases (Pol1, Pol2 and Pol3) and Rev3 contain Fe-S clusters in vivo

a, Wild-type (WT), Gal-NFS1 or Gal-NBP35 cells harboring genomically Myc-tagged Pol1, Pol2, Pol3, were grown in galactose- (gal) or glucose-containing (glc) medium to induce or repress, respectively, production of Nfs1 or Nbp35. After ⁵⁵Fe radiolabelling of cells, polymerases were immunoprecipitated from cell extracts, and bound ⁵⁵Fe was quantified by scintillation counting. TFIIIA-Myc expressed in Gal-NFS1 and WT cells without tagged polymerases (Ctr) served as controls. b, ⁵⁵Fe incorporation into plasmid-encoded HA-Pol3-CTD and HA-Rev3-CTD as in a. Western blots for the cell extracts are presented in Supplementary Figs. 2 and 6 for a and b, respectively. Error bars, s.d. (n ≥ 3).

Figure 2. The Fe-S cluster coordinated by the CysB motif of DNA polymerase CTDs is stabilized by binding of the respective accessory subunits

a, ⁵⁵Fe incorporation into plasmid-encoded HA-Pol3-CTD and cysteine to alanine substitutions thereof (cartoon), with (Pol31↑) or without Pol31 over-expression in wild-type yeast cells. Western blots are for cell extracts with Pol31 over-expression. b, ⁵⁵Fe incorporation into indicated plasmid-encoded polymerase CTDs, with (↑) or without over-expression of the accessory subunits (cartoon) in wild-type yeast cells. Western blots are shown for indicated cell extracts. Full-length blots are presented in Supplementary Fig. 8. Error bars, s.d. (n ≥ 3).

Figure 2. The Fe-S cluster coordinated by the CysB motif of DNA polymerase CTDs is stabilized by binding of the respective accessory subunits

a, ⁵⁵Fe incorporation into plasmid-encoded HA-Pol3-CTD and cysteine to alanine substitutions thereof (cartoon), with (Pol31↑) or without Pol31 over-expression in wild-type yeast cells. Western blots are for cell extracts with Pol31 over-expression. b, ⁵⁵Fe incorporation into indicated plasmid-encoded polymerase CTDs, with (↑) or without over-expression of the accessory subunits (cartoon) in wild-type yeast cells. Western blots are shown for indicated cell extracts. Full-length blots are presented in Supplementary Fig. 8. Error bars, s.d. (n ≥ 3).

Figure 3. Recombinant purified CTDs of Pol1, Pol2, Pol3 and Rev3 harbour a [4Fe-4S] cluster

a, Solubilized inclusion bodies of indicated CTDs obtained after expression in E. coli. b, c, UV-Vis (b) and X-band EPR spectra (c) of purified soluble Pol2-, Pol3- and Rev3-CTDs in absence of chaotropic agents. The inset in (b) shows non-heme iron and acid-labile sulfide contents. Error bars, s.d. (n ≥ 3). Samples in (c) were reduced with 2 mM sodium dithionite (2 min), EPR conditions: 9.458 GHz, 10 K and 2 mW microwave power.

Figure 3. Recombinant purified CTDs of Pol1, Pol2, Pol3 and Rev3 harbour a [4Fe-4S] cluster

a, Solubilized inclusion bodies of indicated CTDs obtained after expression in E. coli. b, c, UV-Vis (b) and X-band EPR spectra (c) of purified soluble Pol2-, Pol3- and Rev3-CTDs in absence of chaotropic agents. The inset in (b) shows non-heme iron and acid-labile sulfide contents. Error bars, s.d. (n ≥ 3). Samples in (c) were reduced with 2 mM sodium dithionite (2 min), EPR conditions: 9.458 GHz, 10 K and 2 mW microwave power.

Figure 4. Functional integrity of purified Pol δ complex depends on binding of an Fe-S cluster to Pol3

Pol δ complex purified from yeast co-expressing wild-type (WT), CysA_MUT (C1012S/C1027S) or CysB_MUT (C1059S/C1074S) Pol3 with Pol31 and Pol32 was analyzed by (a) Coomassie-stained SDS-PAGE, (b) UV-Vis spectroscopy and chemical analysis (inset) or (c) EPR spectroscopy. EPR conditions and treatments with ferricyanide (2 mM) or dithionite are as in Fig. 3c. The full-length gel for a is presented in Supplementary Fig. 11c. Error bars, s.d. (n ≥ 3).

Figure 5. The CysA motif of Pol δ is critical for processive DNA replication with PCNA

a, Alkaline agarose gel electrophoresis of DNA replication products of assay with purified proteins as indicated. A phosphorimaging scan is shown. ΔPIP refers to truncated Pol32 (Supplementary Fig. 11). All complexes contain wild-type Pol31. The right-most lane is a control with WT Pol δ but without PCNA. Size markers are indicated on the left. b, Assay as in a was carried out for 10 min, and with the indicated PCNA concentrations. c, Model for the roles of the PIP and CysA motifs for PCNA interaction with Pol δ in solution (Off DNA), and in assembly with substrate (On DNA).

Figure 5. The CysA motif of Pol δ is critical for processive DNA replication with PCNA

a, Alkaline agarose gel electrophoresis of DNA replication products of assay with purified proteins as indicated. A phosphorimaging scan is shown. ΔPIP refers to truncated Pol32 (Supplementary Fig. 11). All complexes contain wild-type Pol31. The right-most lane is a control with WT Pol δ but without PCNA. Size markers are indicated on the left. b, Assay as in a was carried out for 10 min, and with the indicated PCNA concentrations. c, Model for the roles of the PIP and CysA motifs for PCNA interaction with Pol δ in solution (Off DNA), and in assembly with substrate (On DNA).

Figure 6. Role of mitochondria and Fe-S cluster biogenesis in nuclear DNA replication

Assembly of Fe-S clusters on nuclear replicative DNA polymerases requires Fe-S protein biogenesis machineries located in the mitochondria (Iron-Sulfur Cluster assembly, ISC machinery) and cytosol (Cytosolic Iron-sulfur protein Assembly, CIA machinery) providing an explanation for the essential function of mitochondria for cell viability.