DNA Polymerases at the Eukaryotic Replication Fork Thirty Years after: Connection to Cancer

Abstract

Recent studies on tumor genomes revealed that mutations in genes of replicative DNA polymerases cause a predisposition for cancer by increasing genome instability. The past 10 years have uncovered exciting details about the structure and function of replicative DNA polymerases and the replication fork organization. The principal idea of participation of different polymerases in specific transactions at the fork proposed by Morrison and coauthors 30 years ago and later named "division of labor," remains standing, with an amendment of the broader role of polymerase δ in the replication of both the lagging and leading DNA strands. However, cancer-associated mutations predominantly affect the catalytic subunit of polymerase ε that participates in leading strand DNA synthesis. We analyze how new findings in the DNA replication field help elucidate the polymerase variants' effects on cancer.

Keywords: DNA polymerases; cancer predisposition; mutation rates; proofreading exonucleases; replication fidelity.

Figure 1

Most cancer-associated mutations affect the catalytically active half of POLE. Colored bars represent the main subunits of DNA pols, a catalytic subunit of pol α, POLA1, in light blue; of pol ε, POLE, in yellow; of pol δ, POLD1, in red; and pol ζ, REV3L, in purple. Note that POLE is a tandem of active pol (N-terminal half) and inactive pol (C-terminal half) [5,6]. Evolutionarily conserved motifs characteristic for all exonuclease (exo) domains, are labeled I-V in green and pol domains are labeled I-VI and KxY in red [5,6,7,8,9,10,11,12]. The order of the motifs along all four proteins is the same, but they occupy different parts of the whole protein. For example, REV3L has a very long N-terminal part not related to pols. In POL1, REV3L, and the C-terminal half of POLE, the exonuclease motifs are inactivated during evolution; they are shown in blue. Inactivated pol motifs in the C-terminal half of POLE are shown in black. The key for these and other elements of the pol primary structure is in the left upper quarter of the figure. Rows of circles of different sizes and shades of grey below the catalytic pol subunits represent the number of missense mutations found in tumors along the protein regions in 100 amino acid increments. Variants were collected from the cBioPortal database from a curated non-overlapping collection of tumor genomes (https://www.cbioportal.org/ (cbioportal.org)). A guide explaining the relation between size and intensity of grey to the number of mutations found in the database in the 100 amino acids interval is on the left lower quarter of the figure.

Figure 2

The replication fork seen in 1990 looks almost right in 2020. (A) Schematic representation of the model proposed by Morrison et al. in 1990 [29]. The bidirectional replication starts at the origin, and part of the fork moving to the left is not shown. Primase/Pol α synthesizes short RNA/DNA primers extended by pol ε on the leading strand and by pol δ on the lagging strand. Most of these primers are excised from the newly synthesized DNA [31]. Proofreading exonucleases associated with pol δ and ε have access to 3′-DNA ends on both strands and thus compete to proofread replication errors [24]. The mismatch repair step is not shown for simplicity. (B) The current vision of replication fork. Pol ε does not participate in any transactions on the lagging DNA strand. Pol δ and pol α contribute to the replication of both strands. It is estimated that only 1.5% of DNA synthesized by primase-pol α is retained in newly synthesized DNA in humans [32]. Moreover, 80% of the leading strand is synthesized by pol ε. Pol δ synthesized DNA is at least 18% of the leading [33], and more than 90% of lagging DNA strands [34,35].

Figure 3

Multi-subunit replicative DNA pols. Artistic representations were made based on crystal and cryo-EM structures and models of human primase-pol α [58], yeast pol ε [65,67], yeast pol δ [60], yeast pol ζ [66]. Two latter structures were determined with a truncated third subunit (Table 1), Pol 32, without the C-terminal part, and thus, this part is missing from our drawings. Fe-S cluster () is present in each of the four pol complexes.

Figure 4

Main players at the eukaryotic replication fork. The CMG complex unwinds DNA, primase-Pol α synthesizes short RNA-DNA primers that are extended by pol δ. On the lagging strand, pol δ synthesis is halted when the pol reaches the previous Okazaki fragment. On the leading strand, pol ε takes over and contributes to around 80% of the bulk strand synthesis. Pol δ occasionally proofreads errors made by pol ε [25] and likely continues synthesis on the leading strand thereafter. The coordination of the whole process is likely achieved by interactions of primase-pol α with CMG via Ctf4 (yeast) or AND-1 (humans) [93,94,95,96] and some not precisely mapped pol α interactions with pol δ (dashed black lines) [97,98,99]. Replication stress caused by unusual DNA structures [100], DNA damage or defects in replisome [101,102,103,104] lead to recruitment and patches of synthesis of the fourth member of the B-family, pol ζ [105], along with translesion pols and accessory factors to mitigate replication problems, depending on the nature of replication problems.