Human DNA polymerase delta requires an iron-sulfur cluster for high-fidelity DNA synthesis

Abstract

Replication of eukaryotic genomes relies on the family B DNA polymerases Pol α, Pol δ, and Pol ε. All of these enzymes coordinate an iron-sulfur (FeS) cluster, but the function of this cofactor has remained largely unclear. Here, we show that the FeS cluster in the catalytic subunit of human Pol δ is coordinated by four invariant cysteines of the C-terminal CysB motif. FeS cluster loss causes a partial destabilisation of the four-subunit enzyme, a defect in double-stranded DNA binding, and compromised polymerase and exonuclease activities. Importantly, complex stability, DNA binding, and enzymatic activities are restored in the presence of proliferating cell nuclear antigen. We further show that also more subtle changes to the FeS cluster-binding pocket that do not abolish FeS cluster binding can have repercussions on the distant exonuclease domain and render the enzyme error prone. Our data hence suggest that the FeS cluster in human Pol δ is an important co-factor that despite its C-terminal location has an impact on both DNA polymerase and exonuclease activities, and can influence the fidelity of DNA synthesis.

Figure 1.. Human Pol δ coordinates an FeS cluster within CysB.

(A) Schematic depicting Pol δ and PCNA on a DNA substrate. D2: POLD2, D3: POLD3, D4: POLD4, and CTD: C-terminal domain. (B) Schematic depicting linear arrangement of POLD1 domains (top). The sequence encompassing CysA and CysB from various species is aligned (bottom). Highlighted in orange are the invariant cysteines of CysA and CysB, in red the conserved histidine (H1066) residue within CysB. (C) Quantification of radioactive iron incorporation into wild-type and CysB-variant POLD1, as measured by liquid scintillation counting. Error bars depict standard deviations from three independent experiments. cpm, counts per minute.

Figure S1.. The four invariant cysteines of CysB are required for FeS cluster binding. Related to Fig 1.

(A) Scheme of radioactive iron incorporation assay. Sf9 insect cells are infected with baculoviruses coding for Flag-tagged protein of interest in the presence of radioactive ⁵⁵FeCl₃. Proteins are extracted 48 h postinfection and pulled down using Flag-M2 agarose beads. The beads are washed extensively and subjected to liquid scintillation counting. (B) Quantification of radioactive iron incorporation into the C-terminal domain of wild-type and CysB-variant versions of POLD1, as measured by liquid scintillation counting. (C) Quantification of radioactive iron incorporation into full-length POLD1, as measured by liquid scintillation counting. Error bars depict standard deviations from three independent experiments. cpm, counts per minute.

Figure 2.. FeS cluster loss affects DNA synthesis.

(A) SDS–PAGE showing purified Pol δ in the presence (left) or absence (right) of PCNA. Asterisk denotes baculovirus PCNA that copurifies with Pol δ. (B) Scheme of primer extension assay. Grey circle indicates 5′-fluorescein amidite label. (C, D) Time-course analysis of primer extension with 2 nM of the indicated enzymes in the absence (C) or presence (D) of PCNA. Products were resolved on a denaturing polyacrylamide gel. MW, molecular weight.

Figure S2.. PCNA can restore efficient DNA synthesis of Pol δ-CS. Related to Fig 2.

(A) SDS–PAGE following size-exclusion chromatography of Flag-purified Pol δ (top panel) and Pol δ-CS (bottom panel), both of which were purified in the presence of PCNA. 500 μl of Flag-purified samples were loaded on the column and 300-μl fractions collected. (B) SDS–PAGE showing Flag-PCNA purified from Sf9 insect cells. (C) Primer extension assay with 2 nM of Pol δ-CS in the presence of increasing amounts of PCNA. Products were resolved on a denaturing polyacrylamide gel.

Figure S3.. FeS cluster loss renders Pol δ-CS more sensitive to heat stress. Related to Fig 2.

(A) Scheme of primer extension assay following heat stress. Grey circle indicates 5′-fluorescein amidite label. (B) Following thermal inactivation (Ti) at 55°C for the indicated amounts of time, 2 nM of the indicated enzymes were analysed in a primer extension assay for 10 min and resolved on a denaturing polyacrylamide gel. S: DNA substrate without protein.

Figure 3.. FeS cluster loss affects binding to dsDNA.

(A, B) DNA binding to primer–template junctions was analysed by EMSA with increasing amounts of Pol δ in the absence (A) or presence (B) of PCNA. (C, D) DNA binding to dsDNA was analysed with increasing amounts of Pol δ in the absence (C) or presence (D) of PCNA. (E, F) DNA binding to ssDNA was analysed with increasing amounts of Pol δ in the absence (E) or presence (F) of PCNA. Grey circle indicates 5′-fluorescein amidite label.

Figure 4.. The FeS cluster has an impact on the exonuclease activity of Pol δ.

(A) Scheme of exonuclease assay. Grey circle indicates 5′-fluorescein amidite label. (B, C) Time-course analysis of exonucleolytic degradation with 2 nM of the indicated Pol δ variants in the absence (B) or presence (C) of PCNA. (D) SDS–PAGE showing purified Pol δ3 in the presence (left) or absence (right) of PCNA. MW: molecular weight. (E, F) Time-course analysis of exonucleolytic degradation with 2 nM of the indicated Pol δ3 variants in the absence (E) or presence (F) of PCNA. Products were resolved on a denaturing polyacrylamide gel.

Figure 5.. The FeS cluster impacts on the fidelity of DNA replication.

(A) Scheme of plasmid-based LacZα forward mutation assay. (B) Graphical depiction of percentage of transitions (black), transversions (white), and deletions/insertions (grey) caused by the indicated variants of Pol δ–PCNA complexes.

Figure S4.. The pSJ4-lacZα forward mutation assay. Related to Fig 5.

(A) Gapped pSJ4-lacZα preparation. (B) Detectable mutations in pSJ4-lacZα. (C) Error rate calculation.

Figure S5.. Mutation spectra of Pol δ variants. Related to Fig 5.

(A–E) Plasmids were sequenced from true white colonies following DNA synthesis by Pol δ (WT exo^–) (A), Pol δ (WT exo⁺) (B), Pol δ (CS exo⁺) (C), Pol δ (HY exo⁺) (D), and Pol δ (HW exo⁺) (E), respectively. All gap-fill DNA synthesis reactions had been carried out in the presence of PCNA. Base substitutions are shown above the reference sequence, and deletions (−) and insertions (+) below. Boxes indicate multiple simultaneous alterations.

Figure 6.. The FeS cluster influences the balance between polymerase and exonuclease activities.

(A) Model of human POLD1 structure. N terminus in light grey, exonuclease domain in green, interdomain linker region in yellow, polymerase domain in blue, and C terminus in dark grey. The approximate locations of CysA, CysB, and the interaction sites with PCNA and POLD2 are highlighted in the structure. (B) Graphical representation of primer extension assays in the presence of increasing concentrations of dNTPs. In blue: % of primers extended; in grey: % of primers unextended; and in green: % of primers degraded.

Figure S6.. Variations in the FeS cluster-binding pocket influence the balance between polymerase and exonuclease activities. Related to Fig 6.

(A–D) Primer extension assays with 20 nM of the indicated Pol δ–PCNA complexes in the presence of increasing concentrations of dNTPs. Products were resolved on a denaturing polyacrylamide gel.

Figure 7.. Hypothetical model.

See the Discussion section for details.