Protein-coding gene in the species Homo sapiens
DNA polymerase delta catalytic subunit (DPOD1) is an enzyme that is encoded in the human by the "POLD1" gene, in the DNA polymerase delta complex. DPOD1 is responsible for synthesizing the lagging strand of DNA, and has also been implicated in some activities at the leading strand (Figure 1). The DPOD1 subunit encodes both DNA polymerizing and exonuclease domains, which provide the protein an important second function in proofreading to ensure replication accuracy during DNA synthesis, and in a number of types of replication-linked DNA repair following DNA damage.
Germline mutations impairing activity of POLD1 have been implicated in several types of hereditary cancer, in some sporadic cancers, and in a developmental syndrome of premature aging, Mandibular hypoplasia, Deafness, and Progeroid features and Lipodystrophy (MDPL/MDP syndrome). Studies of POLD1 emphasize the importance of maintaining genomic stability to limit tumorigenesis. It is currently unclear whether the enhanced tumorigenesis associated with "POLD1" defects is the result of increased base substitutions or due to fork collapse and production of DNA double strand breaks (DSBs). Recent reviews have addressed important functions of POLD1.
Discovery.
The first DNA polymerase, DNA polymerase I, was discovered by Arthur Kornberg and his colleagues in 1956, reviewed in. In 1976, Byrnes et al. discovered a third DNA polymerase activity in mammalian cells that was called polymerase delta (δ). It was purified from rabbit erythroid hyperplastic bone marrow and described as a DNA polymerase that possessed an intrinsic 3’ to 5’ exonuclease activity. A 3’-5’ exonuclease proofreading function for DNA polymerases ("E. coli") had first been described 4 years earlier by Kornberg and Brutlag, reviewed in. The human DNA Polδ is a heterotetramer. The four subunits are: (POLD1/ p125), (POLD3/ p66), (POLD2/ p50) and (POLD4/ p12), with the alternative names reflecting the molecular weights expressed in kilodaltons (kDa). The polymerase catalytic subunit was identified as the 125 kDa polypeptide by activity staining in 1991. Several groups independently cloned the human and murine POLD1 cDNAs. Following its purification from various sources including calf thymus, human placenta, and HeLa cells, its activity was implicated in DNA repair.
Gene.
Polymerase (DNA) delta 1, catalytic subunit and "POLD1" are the name and gene symbol approved by the Human Genome Organization (HUGO) Gene Nomenclature Committee (HGNC). "POLD1" is also known as "CDC2, MDPL, POLD," and "CRCS10)," is ~34 kb long and its cytogenetic location is chromosome 19 q13.33. The precise location, in the GRCh38.p2 assembly, is from base pair 50,384,290 to base pair 50,418,018 on chromosome 19. The mouse orthologue maps to mouse chromosome 7. In humans, the major POLD1 transcript (NM_002691.3) contains 27 exons and translates into the 1107 amino acids of the p125 or A subunit. A longer isoform has been reported with a 26 amino acid in-frame insertion after amino acid 592 (NP_001295561.1). A pseudogene (LOC100422453) has been reported on the long arm of chromosome 6. Table 1 provides gene names and chromosomal locations for the various subunits of Polδ in humans, mice, budding yeast ("S. cerevisiae") and fission yeast ("S. pombe").
The "POLD1" gene promoter is regulated via the cell cycle machinery and mRNA expression of "POLD1" reaches a peak in late G1/S phase during DNA replication. The POLD1 promoter is G/C-rich and has no TATA box. The transcription of this GC box-containing promoter is regulated by Sp1 and Sp1-related transcription factors such as Sp3, with their binding mediated via 11-bp repeat binding sequences. The "POLD1" promoter contains an E2F-like sequence located near the major transcription start site. Another regulatory element, the cell cycle element/cell cycle genes homology region (CDE/CHR), located downstream of the start site is important for "POLD1" transcription in G2/M phase by E2F1 and p21 proteins. P53 regulates "POLD1" transcription by indirect p21-dependent activation of a p53-p21-DREAM-CDE/CHR pathway. One study has reported that the p53 tumor suppressor protein competes with Sp1 for binding to the "POLD1" promoter. A microRNA (miR), miR-155, downregulates POLD1 indirectly by suppressing the transcription factor FOXO3a, which has putative binding sites in the "POLD1" promoter (RTMAAYA; response element).
Protein.
POLD1/p125 has a common B-family fold, similar to other DNA polymerases (Polα and ε). Human POLD1/p125 has a putative nuclear localization signal at the N-terminal end (residues 4-19). Residues 304-533 contain the exonuclease domain (Figure 2) while residues 579-974 contain the polymerase domain. The exonuclease domain is a DEDDy-type DnaQ-like domain common to the B-DNA polymerase family. This domain has a beta hairpin structure that helps in switching between the polymerase and exonuclease active sites in case of nucleotide misincorporation.
Motifs A and C, which are the most conserved of the polymerase domain. These have 2 catalytic aspartates, in motif A (DXXLYPS, D602) and motif C (DTDS, D757) that bind calcium at the active site. Motif A has 11 amino acids that are important in nucleotide incorporation and formation of the phosphodiester bond.
Tyrosine Y701 functions similarly to tyrosine Y567 in the RB69 bacteriophage orthologue as the sugar steric gate that prevents ribonucleotide incorporation. An LXCXE motif (711 to 715) mediates binding to pRB during the G1 phase of cell cycle. The polymerase domain also has a highly conserved KKRY motif (residues 806 to 809) which is important for the binding and catalytic function. POLD1 can be targeted to the nucleolus upon acidification via a nucleolar detention sequence (NoDS) motif represented by small sequence motifs dispersed throughout the protein coding region. The C-terminal domain has two conserved cysteine-rich metal-binding motifs (CysA and CysB) (from 1012 and 1083) required for proliferating cell nuclear antigen (PCNA) binding and recruitment of accessory subunits respectively. CysB coordinates an [4Fe-4S] cluster added through Cytosolic Iron-sulfur protein Assembly (CIA), which requires the function of the mitochondrial Iron Sulfur Cluster (ISC) assembly machinery. The maturation process is mediated by the core targeting complex CIA1-CIA2B/FAM96B-MMS19, which interacts with the apoprotein to ensure specific Fe-S cluster insertion.
Binding and association studies have shown that POLD2 is tightly associated with POLD1; POLD3 and POLD2 interact with each other and POLD4 interacts with both POLD1 and POLD2. Polδ heterotetramer reconstituted by coexpression of subunits in Sf9 cells had properties were similar to Polδ purified from the calf thymus, and the complete holoenzyme was very strongly stimulated by PCNA. Numerous studies have shown that while POLD1 possesses both the polymerase and the 3’-5’ exonuclease proofreading activity, the other subunits increase these activities, DNA binding abilities, and functionally important interactions with PCNA and its clamp loader Replication Factor C (RFC). The DNA Polδ holoenzyme is often considered to include PCNA and RFC as well as the four subunits of the polymerase complex (Figure 1).
A number of other studies and screens have identified additional interaction partners relevant to functions in DNA replication and repair. Figure 3 shows a matrix of established and putative interactions during replication and repair which can be further accessed through and. A website at Vanderbilt University provides additional interaction on important POLD1 protein structure and various classes of gene and protein interaction, based on criteria such as co-occurrence in a complex, direct physical interaction, regulatory relationship, and co-expression.
Expression and regulation.
The POLD1/P125 protein is expressed ubiquitously across a panel of human tissues with high levels in the heart and lung tissues. The subcellular localization of POLD1/p125 is predominantly in the nucleus and nucleoplasm.
A reduction in POLD1/p125 has been observed in senescent human skin fibroblasts and in lymphocytes from an elderly population. POLD1/p125 expression is epigenetically regulated in response to DNA damage. Other studies have also shown that POLD1/p125 expression is regulated by miR-155, p53 and by the long non-coding RNA, PVT1. In the presence of DNA damage or replication stress (UV light, methyl methanesulfonate, hydroxyurea or aphidicolin), the POLD4/p12 subunit is rapidly degraded. The catalytic activities of p125 are different whether it is in the heterotetramer (Polδ4, with p12) or in the heterotrimer (Polδ3, without p12). The production of the heterotrimer depends on p12 degradation by the E3 ligase RNF8, a protein involved in DSBs repair and possibly homologous recombination (HR). In addition, the E3 ligase CRL4Cdt2 can degrade POLD4/p12 during normal DNA replication and in the presence of DNA damage. POLD4/p12 can also be degraded by the protease μ-calpain, that is involved in calcium-triggered apoptosis.
POLD1/p125 has a NoDS domain that regulates transport to the nucleolus in response to acidosis. Nucleolar transport requires a direct interaction between the p50 subunit and the WRN protein. During DNA damage response, WRN moves out of the nucleolus and thereby releases Polδ. POLD1/p125 has also been shown to interact with PDIP46/SKAR and LMO2.
Function.
DNA replication.
DNA replication is a highly organized process that involves many enzymes and proteins, including several DNA polymerases. The major replicative activity in S phase of cell cycle depends on three DNA polymerases - Polymerase alpha (Polα), Polymerase delta (Polδ), and Polymerase epsilon (Polε). After initiation of DNA synthesis by Polα, Polδ or Polε execute lagging and leading strand synthesis, respectively. These polymerases maintain a very high fidelity, which is ensured by Watson-Crick base pairing and 3'-exonuclease (or the proofreading) activity. Recent studies have contended that Polδ may synthesize the leading strand. How these polymerases function, in relationship with other factors involved in replication, is of great interest as it likely explains the mutational landscape that they produce when defective. Maintenance of replication fidelity is a fine balance between the unique errors by polymerases δ and ε, the equilibrium between proofreading and MMR, and distinction in ribonucleotide processing between the two strands. Extensive studies in yeast models have shown that mutations in the exonuclease domain of Polδ and Polε homologues can cause a mutator phenotype, reviewed in. The single stranded (ss) DNA synthesized during lagging strand synthesis can be targeted by ss-DNA damaging agents as well as is a selective target for APOBEC mutations. DNA-binding proteins that rapidly reassociate post-replication prevent Polδ from repairing errors produced by Polα in the mature lagging strand. Yeast studies have shown that Polδ can proofread Polε errors on the leading strand.
DNA Repair.
POLD1 activity contributes to multiple evolutionarily conserved DNA repair processes, including Mismatch repair (MMR), Translesion synthesis (TLS), Base excision repair (BER), Nucleotide Excision repair (NER) and double-strand break (DSB) repair. POLD1 mediates the post-incision steps in BER, NER and MMR. Polδ interacts with the MMR machinery to support post-replication proofreading of newly synthesized DNA, with cells bearing mutations that inactivate POLD1 and MMR components experiencing elevated mutation rates. As noted above, a Polδ heterotrimer (Polδ3) becomes the dominant oligomeric form of POLD1 and is active during the presence of DNA damage. Polδ3 is less error-prone than (Polδ4), and can discriminate better between mismatched pairs, associated with better proofreading activity: however, it has reduced ability to bypass some base lesions. Instead, Polδ polymerase switching to the specialized polymerase zeta (Polζ) is important for TLS as the substitution of p125 for the Polζ catalytic subunit, p353, permits better bypass activity. In this process, the highly conserved C-terminal domain (CTD) of POLD1/p125 interacts with the CTD domain of Polζ, and the iron clusters within each CTD mediate interactions involving binding to POLD2 that permit polymerase switching during TLS. Some recent studies suggest that a switch from Polδ to Pol lambda (λ) also supports the TLS and repair of oxidative DNA damage like 7,8-Dihydro-8-oxoguanine lesions.
Depletion of "POLD1" can halt cell cycle at G1 and G2/M phases in human cells. Cell cycle block in these phases typically indicates presence of DNA damage and activation of DNA damage checkpoints. "POLD1" depleted cells are sensitive to inhibition of DNA damage checkpoint kinases ATR and CHK1. In "S. pombe", HR mechanisms could restart stalled replication forks by utilizing Polδ strand synthesis activity, but such nonallelic HR-mediated restart is very error prone potentially leading to increased genomic instability. Polδ structurally and functionally interacts with the WRN protein, and WRN recruits Polδ to the nucleolus. The "WRN" gene is mutated in Werner syndrome (an autosomal recessive disorder) leading to accelerated aging and increased genetic instability. The interaction with WRN increases the processivity of Polδ in a PCNA-independent manner. Through these interactions WRN directly impacts DNA replication-repair and assists in Polδ-mediated synthesis.
Accurate bypass of DNA damage can occur by a recombination-related mechanism involving template switching that employs polymerase δ-dependent DNA synthesis.
Clinical significance.
Cancer.
DNA repair proteins have been shown to be important in human diseases including cancer. For example, germline mutations in DNA repair proteins involved in MMR (MSH2, MLH1, MSH6, and PMS2) have been described in Lynch syndrome (LS), which is characterized by the presence of