Physical and functional interactions of human DNA polymerase eta with PCNA

Abstract

Human DNA polymerase eta (hPoleta) functions in the error-free replication of UV-damaged DNA, and mutations in hPoleta cause cancer-prone syndrome, the variant form of xeroderma pigmentosum. However, in spite of its key role in promoting replication through a variety of distorting DNA lesions, the manner by which hPoleta is targeted to the replication machinery stalled at a lesion site remains unknown. Here, we provide evidence for the physical interaction of hPoleta with proliferating cell nuclear antigen (PCNA) and show that mutations in the PCNA binding motif of hPoleta inactivate this interaction. PCNA, together with replication factor C and replication protein A, stimulates the DNA synthetic activity of hPoleta, and steady-state kinetic studies indicate that this stimulation accrues from an increase in the efficiency of nucleotide insertion resulting from a reduction in the apparent K(m) for the incoming nucleotide.

FIG. 1

PCNA binding motif of hPolη. (A) C-terminal amino acids 699 to 712 of hPolη are aligned with the PCNA binding motifs identified in various PCNA binding proteins. The highly conserved residues are shown in bold. Hs, human; Sc, S. cerevisiae. (B) Mutations made in the PCNA binding motif of hPolη. In the schematic representation of hPolη, the five highly conserved motifs (I to V) shared among different members of the Polη/UmuC/DinB protein family are indicated, and the C₂H₂ motif conserved in the Polη family is shown (20). The amino acid residues present in the extreme C-terminal region are shown; the amino acids highly conserved in the consensus PCNA binding motif are shown in bold. In the hPolη (1-695) mutant protein, the last 18 amino acids from the C terminus were deleted (indicated by an arrow). In the hPolη A⁷⁰⁷-A⁷⁰⁸ mutant protein, the F residues at positions 707 and 708 (indicated by asterisks) were changed to A residues.

FIG. 2

Polη forms a complex with PCNA. Six-His–hPCNA (4 μg) was mixed either with wild-type GST-hPolη protein (lanes 1 to 3 and 13 to 15) or with mutant GST-hPolη A⁷⁰⁷-A⁷⁰⁸ (lanes 4 to 6 and 16 to 18) or GST-hPolη (1-695) (lanes 7 to 9 and 19 to 21) protein (4 μg each). As controls, the same amounts of GST-hPolη (lanes 10 to 12) or six-His–hPCNA (lanes 22 to 24) protein were used alone. After incubation, samples were bound to Ni-NTA (lanes 1 to 12) or glutathione-Sepharose (Seph.) (lanes 13 to 24) beads, followed by washing and elution of the bound proteins by imidazole- or glutathione-containing buffer, respectively. Aliquots of each sample before loading on the beads (L), the flowthrough and wash (F), and the eluted proteins (E) were precipitated by TCA and analyzed on a sodium dodecyl sulfate–12% polyacrylamide gel stained with Coomassie blue. The positions of GST-hPolη and six-His–hPCNA are indicated on the right.

FIG. 3

Stimulation of DNA synthetic activity of hPolη by PCNA. (A) DNA synthesis by the wild-type or PCNA binding site mutant hPolη proteins in the presence or absence of PCNA, RFC, and RPA. The reaction mixtures contained either the wild type hPolη protein (lanes 1 to 8) or the mutant hPolη A⁷⁰⁷-A⁷⁰⁸ (lanes 9 and 10) or hPolη (1-695) (lanes 11 and 12) proteins (10 ng each) along with singly primed M13 single-stranded DNA (25 ng), all four dNTPs (100 μM each), [α-³²P]dATP, PCNA (100 ng), RFC (50 ng), or RPA (250 ng) or combinations of these proteins. No hPolη was added in lane 13. The amount of DNA synthesis is indicated at the bottom as the relative nucleotide (nt) incorporation. HaeIII-digested ϕX174 DNA labeled with polynucleotide kinase is shown on the left as a molecular size marker. (B) Processivity of hPolη in the presence of PCNA, RFC, and RPA. hPolη (10 ng) alone (lanes 1 and 4) or in the presence of PCNA (100 ng), RFC (50 ng), and RPA (250 ng) (lanes 2 and 3) was preincubated with a circular single-stranded M13 template DNA (50 ng) singly primed with a 5′ ³²P-labeled oligonucleotide for 5 min at 37°C. Primer extension reactions were initiated by adding all four dNTPs (500 μM each) (lanes 1 and 2) or all four dNTPs and excess sonicated herring sperm DNA (0.5 mg/ml) as a trap (lanes 3 and 4). After incubation for10 min at 37°C , samples were quenched and run on a 10% polyacrylamide gel. To demonstrate the effectiveness of the trap, hPolη, along with PCNA, RFC, and RPA, was preincubated with the trap DNA and the primer-template substrate before the addition of dNTPs (lane 5).

FIG. 4

PCNA stimulates deoxynucleotide incorporation by hPolη. (A) Steady-state kinetics of deoxynucleotide incorporation opposite an A residue by hPolη in the presence or absence of PCNA, RFC, and RPA in a standing-start reaction. A portion of the DNA substrate is shown at the top. Polη (10 ng) was incubated with a singly primed circular single-stranded M13 (ssM13) DNA substrate (25 ng) and increasing concentrations of a single deoxynucleotide in the absence or presence of PCNA (100 ng), RFC (50 ng), and RPA (250 ng). The nucleotide (nt) incorporation rate was plotted against the dNTP concentration, and the data were fit to the Michaelis-Menten equation describing a hyperbola. The apparent K_m and V_max values were obtained from the fit and used to calculate the efficiency of deoxynucleotide incorporation (V_max/K_m). (B) Steady-state kinetics of deoxynucleotide incorporation opposite a template C residue by hPolη in the presence or absence of PCNA, RFC, and RPA in a running-start reaction. Reactions were carried out as described for panel A, except that each reaction also included the addition of dTTP (15 μM).