The POLD3 subunit of DNA polymerase δ can promote translesion synthesis independently of DNA polymerase ζ

Abstract

The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with normal kinetics. Like its counterpart in yeast, POLD3 is required for fully effective TLS, its loss resulting in hypersensitivity to a variety of DNA damaging agents, a diminished ability to maintain replication fork progression after UV irradiation and a significant decrease in abasic site-induced mutagenesis in the immunoglobulin loci. However, these defects appear to be largely independent of Polζ, suggesting that POLD3 makes a significant contribution to TLS independently of Polζ in DT40 cells. Indeed, combining polη, polζ and pold3 mutations results in synthetic lethality. Additionally, we show in vitro that POLD3 promotes extension beyond an abasic by the Polδ holoenzyme suggesting that while POLD3 is not required for normal replication, it may help Polδ to complete abasic site bypass independently of canonical TLS polymerases.

Figure 1.

pold3 cells exhibit a prolonged S-phase. (A) POLD3 disruption in DT40 cells. The wild-type chicken POLD3 locus from exon 6 to exon 8 was replaced by a puro or bsr selection-marker gene. Targeted loci (middle and bottom) are shown and compared with the relevant chicken POLD3 genomic sequences (top). Solid boxes indicate the position of the exons. Relevant EcoRV sites and the position of the probe used in the Southern blot analysis are indicated. Black arrows indicate the position of primers used for RT-PCR in (C). (B) Disruption of POLD3 was confirmed by Southern blot. (C and D) Depletion of POLD3 mRNA and POLD3 protein in pold3 cells was confirmed by RT-PCR (C) and western blot (D). β-actin was used as an internal control. (E) Relative growth rate plotted for the indicated genotypes. (F) Representative cell-cycle distribution for the indicated genotypes. The top of the box, and the lower left, lower right, and left-most gates correspond to cells in the S, G₁ and G₂/M phases, and the sub-G₁ fraction, respectively. The sub-G₁ fraction represents dying and dead cells. The percentage of cells in each gate is indicated. The box outlined with bold lines corresponds to cells in the late S phase, with the bolded number indicating the percentage of cells. (G) Cells of the indicated genotypes were synchronized at the G₁ phase with elutriation and released into culture. Cell-cycle progression profiles after release are shown.

Figure 2.

The POLD3 deficient cells are sensitive to a wide range of DNA-damaging agents. pold3 cells exhibit hypersensitivity to various types of DNA damage. Cells with the indicated genotype were exposed to the indicated genotoxic agents. The dose of the genotoxic agent is displayed on the x-axis on a linear scale, while the percentage fraction of surviving cells is displayed on the y-axis on a logarithmic scale. Error bars show the SD of mean for three independent assays.

Figure 3.

The important role of POLD3 in TLS past abasic sites during Ig V_λ hypermutation. (A) Ig Vλ segments isolated from indicated cells, clonally expanded for two weeks. Horizontal lines represent the rearranged Ig Vλ (450 bp), with hypermutation (red (gray in print) lollipop shapes), gene conversion (horizontal bars), single-nucleotide substitutions that could be the result of hypermutation or gene conversion (vertical bars) and single-base deletion (boxes) determined as described previously (34,35). More than three clones were expanded for two weeks and analyzed for each dataset. (B) The rates of gene conversion (GC) and hypermutation (PM) are indicated with standard error. (C) Pattern of point mutation in wild-type, pold3 and polζ/polη cells. Tables showing the pattern of mutation in each line, given a percentage of mutations observed. (D) Frequency of mutagenic base insertion of C, T or A opposite C on either strand, corresponding to mutation from C to G, A and T, respectively. The size of the pie charts reflects the frequency of overall point mutation within mutated sequences, while the segments reflect the relative use of C, T or A in bypass.

Figure 4.

Synthetic lethality of pold3 and polζ/polη. (A) Western blot for probing existence of POLD3 protein was shown. Protein samples from indicated cells were analyzed. β-actin was analyzed as a loading control. (B) Growth curves of the indicated cells. The transcription of tet-POLD3 was active without doxycycline (−Dox) and inhibited upon addition of doxycycline (+Dox).

Figure 5.

POLD3 but not Polζ is required to maintain replication fork progression on UV damaged DNA. (A) Intact postreplicative gap filling in pold3 cells. Indicated cells without UV (upper panel) or with UV (5 J/m²) (lower panel) were pulse-labeled with [methyl-¹⁴C] thymidine, then incubated a further in fresh medium containing 10 μM unlabeled thymidine for the indicated time. Samples were separated on 5–20% alkaline sucrose gradient sedimentation. (B) Representative image showing stained DNA fibres. DT40 cells were labelled sequentially with IdU and CldU with or without UV treatment after IdU labeling. (C) The data for cells carrying the indicated genotypes was plotted as a cumulative percentage (y-axis) of forks at each ratio (x-axis). The P-values of the Kolmogorov–Smirnov test for ratio distribution of each mutant for UV compared to wild-type are indicated. n.s.: not significant.

Figure 6.

POLD3 is required for efficient Polδ-dependent TLS past abasic sites in vitro. (A and B) DNA synthesis reactions carried out with the indicated Polδ holoenzymes (2 nM each) on template and primer strands, which are schematically shown on the left. The 49-nucleotides template strands carry a dC (A) or abasic site (B) at the 25th nucleotide from the 3′ end. The 5′ end of the primer was ³²P labeled as shown by a star. The DNA synthesis was done the presence of NaCl (8 mM), MgCl₂ (7mM), PCNA (50 nM) and dNTP (10 μM), a concentration which reflects that in human cycling cells (37). After incubation at 37°C for 10 min, reaction products were separated on a 15.6% polyacrylamide gel containing 7 M urea and analyzed using a PhosphoImager. (B) The bands shown by −1 and +0 represent the DNA strand having stopped its extension one nucleotide before and at the abasic site, respectively. (C) The amounts of extension product (49 nucleotides) relative to the amount of input primer is shown as percentage. The experiment was performed at least three times, and averages are presented with SD and P-values. (D) Degradation and extension of the primer carrying a dA opposite the abasic site of the shown template DNA strand with the indicated Polδ holoenzymes (2 nM each) was examined at 15 min. The reaction was carried out with the indicated concentrations of dNTP. Less than 0.1 μm concentrations of dNTP significantly enhanced the proofreading exonuclease over polymerase activity. (E) Kinetics of the primer degradation in the absence of deoxynucleotides was examined. (Left) Reaction was carried out with 2 nM of the indicated Polδ holoenzymes for the indicated time. (Right) Reaction was carried out with the indicated concentrations of Polδ holoenzymes for 15 min.