POLD3 as Controller of Replicative DNA Repair

Abstract

Multiple modes of DNA repair need DNA synthesis by DNA polymerase enzymes. The eukaryotic B-family DNA polymerase complexes delta (Polδ) and zeta (Polζ) help to repair DNA strand breaks when primed by homologous recombination or single-strand DNA annealing. DNA synthesis by Polδ and Polζ is mutagenic, but is needed for the survival of cells in the presence of DNA strand breaks. The POLD3 subunit of Polδ and Polζ is at the heart of DNA repair by recombination, by modulating polymerase functions and interacting with other DNA repair proteins. We provide the background to POLD3 discovery, investigate its structure, as well as function in cells. We highlight unexplored structural aspects of POLD3 and new biochemical data that will help to understand the pivotal role of POLD3 in DNA repair and mutagenesis in eukaryotes, and its impact on human health.

Keywords: DNA repair; DNA replication; POLD3; Polδ; Polζ; helicase; histone.

Figure 1

Pan-cancer analysis of POLD3 expression in tumor tissue (pink) compared with non-tumor tissue (blue). The levels of gene expression are upregulated in glioblastoma multiforme (GBM), glioblastoma and low-grade glioma (GBMLGG), brain lower grade glioma (LGG), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), esophageal carcinoma (ESCA), stomach and esophageal carcinoma (STES), colon adenocarcinoma (COAD), colon adenocarcinoma/rectum adenocarcinoma esophageal carcinoma (COADREAD), stomach adenocarcinoma (STAD), head and neck squamous cell carcinoma (HNSC), lung squamous cell carcinoma (LUSC), liver hepatocellular carcinoma (LIHC), high-risk Wilms tumor (WT), skin cutaneous melanoma (SKCM), pancreatic adenocarcinoma (PAAD), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (LAML) and cholangiocarcinoma (CHOL); and downregulated in kidney renal papillary cell carcinoma (KIRP), kidney renal clear cell carcinoma (KIRC), kidney chromophobe (KICH), pan-kidney cohort (KICH + KIRC + KIRP, KIPAN), prostate adenocarcinoma (PRAD), thyroid carcinoma (THCA), testicular germ cell tumors (TGCT), uterine carcinosarcoma (UCS), and adrenocortical carcinoma (ACC). ** p < 0.01. *** p < 0.001. **** p < 0.0001. Data were collected from the TCGA and GTEx, processed using Sangerbox3.0 [19].

Figure 2

Timeline of POLD3 proteins studies. Nomenclature is described in the main text. We also refer to a timeline for DNA polymerases discoveries and studies provided by the Woodgate group: https://www.nichd.nih.gov/research/atNICHD/Investigators/woodgate/research/DNA-polymerases (accessed on 21 September 2024).

Figure 3

POLD3 in Polδ and Polζ complexes. (A,B) Experimentally determined interactions between subunits within Polδ and Polζ shown as solid arrows, although with a questionable interaction of POLD1 and POLD3. More detail about the experimental data can be found in Section 4 of the text; (C,D) Structures of human Polδ (PDB: 6TNY) and S. cerevisiae Polζ (PDB: 6V8P). The polymerase/nuclease catalytic subunits are in grey—POLD1 in Polδ and Rev3 in Polζ. POLD2 is yellow, and the partial structure of POLD3 (see detail in main text) is blue. POLD4 in Polδ is pink, and the Rev7 dimer of Polζ is green; (E) The completely resolved structure of human POLD2 (yellow) with its interaction to POLD3 (blue), POLD4 (pink) and two α-helixes of POLD1 (grey). The POLD2 CysA and CysB motifs are highlighted in red, and the iron–sulfur cluster of CysB (green) is thought to regulate POLD1 catalytic function; (F) Electrostatic surface of the POLD2–POLD3 interaction in human Polδ (PDB 6TNY), with negatively charged POLD2 and the β-sheet 447-CQPISFSG-454 in red, and positively charged POLD3 residues and β-sheet 73-SCHKVAVV-80 in blue; (G) POLD3 isoleucine-10 (ILE 10) highlighted in a clinical case report [11], may interact with POLD3 tyrosine-44 (TYR 44), a proposed substrate for phosphorylation. The minimum distance between the elements in two residues is 3.61Å. Images and evaluation of element distance were produced using ChimeraX from PDB 6TNY.

Figure 4

POLD3 has an N-terminus wHTH fold that is conserved across eukaryotes (see also Figure 3), and more C-terminal IDPRs containing some conserved motifs. (A) POLD3 alignment from 12 organisms framing in red squares the conserved amino acid motifs that have been studied: DNA polymerase interaction motif (DPIM); nuclear localization sequence (NLS), which seems to be present only in vertebrates and invertebrates but not in the fungi; Rev1 interaction region (RIR) typically formed by two hydrophobic phenylalanine amino acids; and PCNA interacting protein (PIP) motif. Those motifs (yellow) in POLD3 IDPR (blue) on a predicted POLD3 structure by Alphafold3, the wHTH fold of POLD3 is marked in grey. The organisms used were as follows: Homo sapiens (human), Bos taurus (cow), Mus musculus (mouse), Phascolarctos cinereus (koala), Gallus gallus (chicken,), Xenopus laevis (african clawed frog), Caenorhabditis elegans (roundworm), Drosophila melanogaster (fruit fly), Schizosaccharomyces pombe (fission yeast), Saccharomyces cerevisiae (budding yeast), Chaetomium thermophilum (thermophilic fungus) and Candida albicans (pathogenic yeast) for the sequence alignment. (B) Summary of the motifs of POLD3 proteins: The N-terminus resolved wHTH domain that is crucial for interaction with POLD2 (grey), and the unstructured C-terminus IDPR (blue), which contains the Rev1 interaction region (RIR), two nuclear localization signals (NLS), and a site containing a cluster of post-translational modifications (PTMs).

Figure 5

POLD3 is necessary for DNA repair by break-induced replication. (A) Shows DNA strand invasion catalyzed by a recombinase enzyme such as RAD51 (pink, bean-shape icon), which primes new DNA synthesis by Polδ (POLD3 colored blue, other subunits colored in grey). Mutations (red stars) are caused by DNA synthesis in this D-loop context, but the cell has survived a potentially lethal chromosome break; (B) A possible DNA polymerase switch from Polδ to Polζ. It is thought that during mutagenic DNA synthesis, in this context, the polymerase switches between Polδ and Polζ. This may come about by the PCNA (purple) interaction with POLD3 (blue)–POLD2 (yellow) core complex via the PIP motif at POLD3 C-terminus, with POLD1 (grey) and POLD4 (red) disassociating from the complex. The REV3L (grey)-REV7 heterodimer (green) forms a complex with POLD2–POLD3, thus Polζ proceeds DNA replication. This mechanism is likely one of the factors that causes mutagenesis during BIR.

Figure 6

POLD3 exerts multiple functions during DNA replication. In this process, MCM2 of the MCM complex transfers parental histones H3-H4 to POLD3, which are then passed to Pol1 of Polα. In addition, POLD3 can independently interact with newly synthesized H3-H4. In parallel with histone disposal, POLD3 also regulates the catalytic function of Polδ/POLD1 and interacts/coordinates with DNA repair helicases—see main text such—highlighted as double arrows.