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Review
. 2008:82:101-45.
doi: 10.1016/S0079-6603(08)00004-4.

DNA polymerase epsilon: a polymerase of unusual size (and complexity)

Zachary F Pursell  1 , Thomas A Kunkel
Affiliations
Review

DNA polymerase epsilon: a polymerase of unusual size (and complexity)

Zachary F Pursell et al. Prog Nucleic Acid Res Mol Biol. 2008.
No abstract available

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Figures

Figure 1
Figure 1. Pol ε: At the Intersection of Complex and Diverse Cellular Processes
Pol ε is involved in a wide array of diverse cellular processes. Its involvement is depicted here with the direction and size of the arrows meant to reflect the direction of influence and relative importance of its involvement in each process, respectively. By functioning as the leading strand polymerase in normal replication, a large arrow points from DNA replication towards Pol ε (see text in section E). However, some data suggest that, while important for normal replication, the essential function of Pol ε lies in its role as a checkpoint sensor during replication (see section F), influencing both replication itself as well as cell cycle progression. Thus large arrows point away from Pol ε toward these processes. Pol ε is also implicated in the repair of damaged DNA, though the degree to which it operates in specialized repair pathways remains unclear (see sections I and J). Pol ε contributes to altering chromatin status, typically by promoting a silenced state either transiently or in a heritable, epigenetic manner (see sections G and H), thus arrows point from Pol ε toward these processes. Additionally, alterations to chromatin or epigenetic modifications may target Pol ε to these regions, thus arrows point from these processes toward Pol ε. The interaction of Pol ε with other factors (see section C) as well as the intrinsic fidelity and other biochemical properties of Pol ε (see section D) play important roles throughout each of these processes. Proper cell cycle progression, DNA damage repair and DNA replication, and possibly chromatin and epigenetic states as well, serve the overall goal of maintaining genome stability.
Figure 2
Figure 2. Pol ε Catalytic Subunit
(A) A schematic of the Pol ε catalytic subunit. Conserved motifs in the exonuclease and polymerase domains are shown in yellow, with the C-terminal protein-protein interaction region in red. DEAD-box cleavage sites in human Pol ε are shown as black arrows. (B) The structure of the Pol ε homologue RB69 DNA polymerase complexed with an incoming (correct) dTTP and primer-template DNA is shown using coordinates from PDB accession number 1IG9 (11). The fingers, palm, thumb, and exonuclease domains are shown in blue, purple, green, and brown, respectively. The duplex DNA is yellow and dTTP shown at the polymerase active site is red. The light blue spheres represent the divalent metal ions in the polymerase and exonuclease active sites. (C) Alignment of the amino acid sequences of conserved polymerase motifs A, B, and C from Pol ε and other representative B family polymerases. Conserved catalytic aspartate residues are shown in the black boxes. The conserved motif A methionine that differs between Pol ε and the other B family polymerases is shown in a gray box with a magenta star. Pol2, p261, and cdc20 are Pol ε from S. cerevisiae, H. sapiens, and S. pombe, respectively. Pol1 and Pol3 are S. cerevisiae pols α and δ, respectively. RB69 and ϕ29 are bacteriophage DNA polymerases. (D) Ribbon diagram depicting an overlay of the structures of polymerase motifs A, B, and C from three B family DNA polymerases. Coordinates from PDB accession numbers 2PYL (ϕ29 pol, cyan), 1IG9 (RB69 pol, magenta), 1TGO (Tgo pol, gray), and 1QQC (D.tok pol, yellow) were used to align the structures with PyMol. The conserved Leu/Met that was altered to generate the mutator alleles described in the text is shown as a magenta star in the RB69 Pol structure. (E) Alignment of the amino acid sequences of conserved exonuclease motifs I, II, and III from Pol ε and other B family polymerases. Conserved catalytic carboxylates are shown in black boxes. DNA polymerases are as in (C).
Figure 2
Figure 2. Pol ε Catalytic Subunit
(A) A schematic of the Pol ε catalytic subunit. Conserved motifs in the exonuclease and polymerase domains are shown in yellow, with the C-terminal protein-protein interaction region in red. DEAD-box cleavage sites in human Pol ε are shown as black arrows. (B) The structure of the Pol ε homologue RB69 DNA polymerase complexed with an incoming (correct) dTTP and primer-template DNA is shown using coordinates from PDB accession number 1IG9 (11). The fingers, palm, thumb, and exonuclease domains are shown in blue, purple, green, and brown, respectively. The duplex DNA is yellow and dTTP shown at the polymerase active site is red. The light blue spheres represent the divalent metal ions in the polymerase and exonuclease active sites. (C) Alignment of the amino acid sequences of conserved polymerase motifs A, B, and C from Pol ε and other representative B family polymerases. Conserved catalytic aspartate residues are shown in the black boxes. The conserved motif A methionine that differs between Pol ε and the other B family polymerases is shown in a gray box with a magenta star. Pol2, p261, and cdc20 are Pol ε from S. cerevisiae, H. sapiens, and S. pombe, respectively. Pol1 and Pol3 are S. cerevisiae pols α and δ, respectively. RB69 and ϕ29 are bacteriophage DNA polymerases. (D) Ribbon diagram depicting an overlay of the structures of polymerase motifs A, B, and C from three B family DNA polymerases. Coordinates from PDB accession numbers 2PYL (ϕ29 pol, cyan), 1IG9 (RB69 pol, magenta), 1TGO (Tgo pol, gray), and 1QQC (D.tok pol, yellow) were used to align the structures with PyMol. The conserved Leu/Met that was altered to generate the mutator alleles described in the text is shown as a magenta star in the RB69 Pol structure. (E) Alignment of the amino acid sequences of conserved exonuclease motifs I, II, and III from Pol ε and other B family polymerases. Conserved catalytic carboxylates are shown in black boxes. DNA polymerases are as in (C).
Figure 3
Figure 3. Pol ε Holoenzyme
(A) Schematic representation of each of the three Pol ε accessory subunits. Sites of known in vitro and potential in vivo phosphorylation (17) are shown as red and black circles, respectively. Histone-fold motifs are shown in orange. (B) Cartoon of four-subunit Pol ε holoenzyme. Each subunit is drawn approximately to scale, based on its predicted molecular weight. Human (and yeast) gene names are indicated next to each subunit. (C) Cryo-EM structure of four-subunit yeast Pol ε (from (23). The open conformation is shown on the left, while the right depicts a model of a closed conformation with duplex DNA bound. This image is reprinted from (23) with permission from the authors.
Figure 4
Figure 4. Nascent Base Pair Binding Pocket of A B Family Polymerase
Surface representation of the nascent base pair and several amino acids in RB69 Pol that form the DNA minor groove edge of the binding pocket at the polymerase active site (Adapted from (212) with the author’s permission). Met644 in yeast Pol ε (in parentheses) aligns with Leu415 in RB69 Pol (green). The adjacent Tyr416 in RB69 Pol aligns with Tyr869 in yeast Pol α, which, when substituted with alanine, results in a mutator phenotype.
Figure 5
Figure 5. Model of A Eukaryotic Replication Fork
This model is based on the currently favored hypothesis that Pol ε is primarily responsible for leading strand synthesis, shown in blue, and Pol δ is primarily responsible for lagging strand synthesis, shown in green. Pol α-primase cooperates with Pol δ to conduct lagging strand synthesis, with the initiating RNA primers shown in red. RPA heterotrimers are shown in violet. The CMG replicative helicase is shown as a heterohexameric MCM complex (orange) associated with the GINS complex (green) and Cdc45 (light red).
Figure 6
Figure 6. Model of Histone-Fold Subunits-DNA Interaction
Shown is a structural alignment of the heterodimer DmCHRAC-14/DmCHRAC-16 (shown in red/blue, respectively) from (138) with the DNA-bound heterodimer of histone H2A–H2B (magenta/light blue, respectively) from (213). DmCHRAC-14 is the same as DmPol ε-p17. The blue circle indicates where the KKK→AAA triple mutant of Dpb3 that results in loss of DNA-binding and telomeric silencing (52) maps to the structure. The green circle represents where the S/T→KK mutant in Dpb4 that partially suppresses the KKK→AAA Dpb3 mutant maps to in the structure. The α1, α2, α3, and αC helices on each HFM subunit are indicated. N- and C-terminal ends of histones are indicated.
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