POLD1: Central mediator of DNA replication and repair, and implication in cancer and other pathologies

Abstract

The evolutionarily conserved human polymerase delta (POLD1) gene encodes the large p125 subunit which provides the essential catalytic activities of polymerase δ (Polδ), mediated by 5'-3' DNA polymerase and 3'-5' exonuclease moieties. POLD1 associates with three smaller subunits (POLD2, POLD3, POLD4), which together with Replication Factor C and Proliferating Nuclear Cell Antigen constitute the polymerase holoenzyme. Polδ function is essential for replication, with a primary role as the replicase for the lagging strand. Polδ also has an important proofreading ability conferred by the exonuclease activity, which is critical for ensuring replicative fidelity, but also serves to repair DNA lesions arising as a result of exposure to mutagens. Polδ has been shown to be important for multiple forms of DNA repair, including nucleotide excision repair, double strand break repair, base excision repair, and mismatch repair. A growing number of studies in the past decade have linked germline and sporadic mutations in POLD1 and the other subunits of Polδ with human pathologies. Mutations in Polδ in mice and humans lead to genomic instability, mutator phenotype and tumorigenesis. The advent of genome sequencing techniques has identified damaging mutations in the proofreading domain of POLD1 as the underlying cause of some inherited cancers, and suggested that mutations in POLD1 may influence therapeutic management. In addition, mutations in POLD1 have been identified in the developmental disorders of mandibular hypoplasia, deafness, progeroid features and lipodystrophy and atypical Werner syndrome, while changes in expression or activity of POLD1 have been linked to senescence and aging. Intriguingly, some recent evidence suggests that POLD1 function may also be altered in diabetes. We provide an overview of critical Polδ activities in the context of these pathologic conditions.

Keywords: DNA damage response; Hypermutation; MDPL syndrome; POLD1; Polymerase delta; Replication; p125.

Fig. 1

POLD1 gene and promoter structure. A. Splicing structure. Schematic representation of the number and sizes of exons and introns of transcript NM_002691.3. The information was extracted from GeneTable on the NCBI website (http://www.ncbi.nlm.nih.gov/gene/5424?report=gene_table). The coding sequence is from nucleotides 70 to 3393 of the spliced mRNA. The encoded protein NP_002682.2 has 1107 amino acids. The first amino acid encoded by each exon is indicated. The residues overlapping a splice site are underlined. B. Schematic representation of the POLD1 promoter structure. The two 11 bp repeats (underlined) were identified by Zhao and Chang (1997). The p53 binding site (in blue) has 17 of 20 nucleotides matching the canonical site; a 5 bp spacer harbors a Sp1 binding site between its two halves (black double arrow). This site was identified as functional in Li and Lee (2001). An E2F binding site overlaps the 3′ end of the motif (green double arrow). The CDE/CHR element, important for cell cycle regulation, was identified and functionally analyzed in Song et al. (2009). The forkhead response element may be involved in the regulation of expression by miR-155 (Czochor et al., 2016).

Fig. 2

A simplified view of the function of Polδ at the DNA replication fork and in response to damaged DNA. A. The Polδ complex (p125, p66, p50 and p12) associates with replication forks. Exo marks the exonuclease domain of p125 and Polε. The MCM helicase (light lime green) drives the replication fork forward. The single-stranded regions are coated with the single-stranded binding protein, replication protein A (RPA) (pink). Polα is bound to a primase, which initiates synthesis of lagging strand (black line) by producing an RNA primer which is then elongated first by Polα, then by Polδ. Polε is positioned on the leading strand (orange line). GINS (go-ichi-ni-san comprising of four related subunits of the complex Sld5, Psf1, Psf2 and Psf3) (Lujan et al., 2016a, Lujan et al., 2016b) interacts with Polε to initiate DNA synthesis. Some data suggest a role of the Polδ complex in the leading strand synthesis. Both polymerases use PCNA (proliferating cell nuclear antigen; green rings) as a sliding clamp. The RFC (replication factor C) complex in conjunction with RPA loads PCNA onto the DNA. PCNA-loading typically requires ATP, although ATP-independent mechanisms have been suggested (Burgers and Yoder, 1993, Chen et al., 2009). As replication progresses, nucleosomes are displaced and single-stranded DNA is bound by RPA. As the lagging strand is synthesized in short fragments, Okazaki fragments, ligases (ligase I) are used to seal gaps. Replication errors created by the polymerases (indicated as open black triangle on the newly synthesized leading strand), can be corrected by post-replication mismatch repair (MMR). As recently reviewed by Johansson and Dixon (2013) and discussed by others (Shamoo and Steitz, 1999), it has been difficult to isolate intact replisomes. Work from Georgescu et al. suggests that the eukaryotic replisome is asymmetric in its architecture with Polε on the leading strand and Polδ on the lagging strand (Georgescu et al., 2014, Georgescu et al., 2015, Zhang and O'Donnell, 2016). Additional details on the replisome architecture, including relative positioning of Polδ and PCNA, have been recently reviewed (Zhang and O'Donnell, 2016). B. Multiple forms of DNA damage can activate an intra-S phase checkpoint. This recruits and activates ATR to the site of DNA damage, triggering downstream DNA damage response signaling. During this process, Polδ is recruited to repair foci. P12 is ubiquitinated (black circles, Ub) and degraded by the proteasome, which leads to the conversion of Polδ4 to the Polδ3 complex, which has altered catalytic activity. It is possible to rapidly exchange between the Polδ3 and Polδ4 complexes (two-way green arrow) (Lee et al., 2012).

Fig. 3

p125/POLD1 protein structure. A. Schematic domain structure and motif sites in human p125/POLD1. The exonuclease and polymerase domains are shown in green and blue respectively. Conserved motifs are shown in darker shades. The ExoI–III motifs, and motifs A–C are highly conserved in the B-family of polymerases. ExoIV and V are conserved between Polδ and Pol ε (Hansen et al., 2015). The LXCXE motif was reported by Krucher et al. (2000). The nucleolar detention sequence (NoDS) defined by the motif RR(I/L)XXXR and a least two hydrophobic triplets with leucine as first residue and leucine or valine as last residue is represented by the amino acid RRLLIDR and nine hydrophobic triplets (Mekhail et al., 2007). In p125, this is represented by the amino acids 849–851 (RRL) and nine hydrophobic triplets (3 LAL, 2 LGL, 2 LAV, 1 LQV, 1 LFV starting at leucine residues 38, 188, 340, 353, 460, 508, 630, 691 and 943) (Mekhail et al., 2007, Audas et al., 2012). The cysteine motifs CysA and CysB located in C-terminal are a Zn-binding site and a Fe–S cluster respectively (Netz et al., 2012). The figure also shows the nuclear localization signal (NLS) in the amino-terminus (pink). B. Enlarged schematic of the exonuclease (green) and polymerase (blue) domains, representing mutations discussed in text. Black denotes germline mutations, orange denotes somatic mutations and red denotes mutations detected in human cell lines.

Fig. 4

Frequency of somatic mutations in different cancers extracted from cancer studies in the TCGA (The Cancer Genome Atlas) (data retrieval date March 30th 2016). Abbreviations used on the graph are CRC, colorectal cancer; lung AD, lung adenocarcinoma; lung SC, lung squamous carcinoma; ccRCC, clear cell renal cell carcinoma; uterine CEC, uterine corpus endometrial carcinoma.