DNA Polymerase ε: A Polymerase of Unusual Size (and Complexity)
Introduction
Shortly after Kornberg et al. identified the first DNA polymerase in the mid-twentieth century (1), the first eukaryotic DNA polymerase was discovered (2), and was eventually named DNA Pol α. Pol α was initially believed to be the sole polymerase responsible for eukaryotic DNA replication, but that view changed two decades later with the discovery of a second replicative polymerase, Pol δ (3). Although the catalytic subunit of Pol δ is 125 kDa, soon after its discovery even larger polymerases were purified that had somewhat similar properties and were therefore variously called Pol δII, Pol δ*, and big Pol δ (4., 5., 6.). However, biochemical studies eventually led to the realization that these larger enzyme forms were actually a distinct polymerase, Pol ε (7). Although much of the early work on Pol ε centered on its initially defined role in mammalian DNA repair, by the early 1990s, the gene encoding Pol ε was cloned (8., 9.), making possible the many genetic and biochemical studies that are the subject of this chapter. From these studies, we now know that Pol ε is involved in several processes that are central to maintaining the stability of the eukaryotic nuclear genome (Fig. 1). These include DNA replication, repair of DNA damage, control of cell cycle progression, chromatin remodeling, and epigenetic regulation of the stable transfer of information from mother to daughter cells. Here we consider the evidence supporting these many functions for Pol ε, and in doing so point out that a great deal remains to be learned about this large and complex polymerase.
Section snippets
The Catalytic Subunit
DNA pol ε is a member of the B family of DNA polymerases that share sequence homology with the catalytic subunit of bacterial Pol II, the product of the Escherichia coli polB gene. The open reading frame for the POLE1 gene encoding the Pol ε catalytic subunit (Fig. 2A) is among the longest of the many known eukaryotic polymerases (10), rivaled only by those of its B family sibling Pol ζ and the A family enzyme, Pol θ. The catalytic subunits of human (Fig. 2A) and yeast Pol ε contain 2286 and
Physical and Functional Interactions of Pol ε
In addition to interactions among the four subunits of the holoenzyme, Pol ε has also been shown to interact with other proteins (Table II). One interacting partner is yeast Dpb11 (25), known as TopBP1 in humans and variously known as Rad4, Cut5, and Mus101 in other species. Dpb11 is a BRCT repeat-containing protein initially identified as a multicopy suppressor of temperature-sensitive mutants in the C-terminus of Pol2 and of mutants in Dpb2 (26) that is loaded onto origins after
Polymerization
Pol ε catalyzes DNA template-dependent DNA synthesis by a phosphoryl transfer reaction involving nucleophilic attack by the 3′ hydroxyl of the primer terminus on the α-phosphate of the incoming deoxynucleoside triphosphate (dNTP). The products of this reaction are pyrophosphate and a DNA chain increased in length by one nucleotide. The catalytic mechanism is conserved among DNA polymerases (41). It begins with binding of a primer template to the polymerase. The primer terminus is bound at the
Pol ε in DNA Replication
In order for eukaryotic cells to divide and pass along their genetic complement to each daughter cell, the entire genome must be replicated accurately once, and only once, per division. This is an ordered process whereby origins of replication, ranging from well-defined 125 bp ARS sequences in Saccharomyces cerevisiae to relatively poorly understood zones of replication of many kb in mammalian cells, are licensed by the binding of pre-RC components, including the ORC complex, during the G1 phase
Cell Cycle Progression and Replication
The cell cycle is an orderly progression of events that allows the genome to be completely duplicated prior to the onset of mitosis. Unrepaired DNA damage sustained at any point in the cell cycle can disrupt this progression. DNA repair and checkpoint activation are the two means by which cells can avoid the potentially deleterious uncoupling of normal cell cycle progression. Checkpoints are cellular pathways that involve slowing or blocking cell cycle progression to allow time for repair of
Pol ε Influence on Gene Silencing
Heterochromatic DNA is transcriptionally silent, and silenced regions are able to epigenetically influence neighboring regions through the use of Sir proteins, including the NAD+-deacetylase Sir2. Silencing involves establishment, maintenance, and inheritance of the silent state (112). Budding yeast establish, maintain, and propagate three different silent regions through differential involvement of the Sir proteins: rDNA, silent mating-type loci, and telomeres. The rDNA exists in an array of
Chromatin Remodeling
In order to package the entire genome into the nucleus and to control gene expression, DNA in eukaryotic cells is organized into chromatin (126). At the nucleic acid level, 146 bp of DNA is wrapped in two turns around a histone octamer composed of a core of two H3/H4 histone dimers flanked by two pairs of H2A/H2B histone dimers forming the nucleosome. These nucleosomes are separated by histone-free linker DNA and are arrayed along the DNA, which coils into 30 nm helices known as chromatin fibers
The Roles of Pol ε in Excision Repair of DNA Damage
Much of the early work implicating Pol ε in DNA repair synthesis was based in large part on studies making use of the DNA polymerase inhibitor, aphidicolin (5., 146.). This was done primarily to distinguish aphidicolin-sensitive DNA synthesis from aphidicolin-insensitive DNA polymerases like Pol β. Unfortunately, aphidicolin inhibits both Pol δ and Pol ε, making distinctions between the two using this strategy difficult. Even today, it remains difficult to distinguish the involvement of the two
Double-Strand Break Repair
Double-strand breaks (DSB) in DNA are among the most toxic of lesions; even one DSB can be lethal (167). DSBs arise through a number of different endogenous and exogenous sources including γ-irradiation, mating type switching, immunoglobulin rearrangement, crossing over during meiosis, and stalled replication forks [reviewed in (147)]. Two major types of repair abrogate the deleterious effects of DSBs, nonhomologous end-joining, and homologous recombination (168., 169.). The latter can be
Catalytic Subunit
Schizosaccharomyces pombe Pol ε, or cdc20, was initially described as a mutant allele defective in the initiation of DNA replication (187) and with reduced sporulation efficiency (188). Thermosensitive mutants in cdc20, along with mutants of DNA polymerase δ and MCM4, undergo mitosis prior to completing replication (189), much like the S. cerevisiae C-terminal truncation mutants, linking Pol ε to the DNA damage checkpoint in fission yeast. When the cdc20 gene was cloned and found to encode Pol ε
Xenopus Pol ε
Understanding Pol ε function has been facilitated using the powerful cell-free replication system of Xenopus (81). Immunodepleting Pol ε from Xenopus extracts leads to a defect in bulk DNA synthesis (81) that is complemented by recombinant Xpol ε (195). This defect is more pronounced in extracts depleted of Pol δ, which also accumulated large amounts of ssDNA (85), consistent with a role for Pol δ in lagging strand synthesis. As in S. cerevisiae (196), Xenopus Pol ε binds to chromatin in a
Concluding Remarks
Pol ε plays a central role in replication fork establishment, progression, and maintenance of fork stability (Fig. 1). In addition, Pol ε plays important roles in the establishment and maintenance of a silenced chromatin state, the repair of DNA base damage, and the restart of stalled replication forks. Each of these processes must occur faithfully and in a regulated manner in order to allow duplication of the eukaryotic genome and stable transmission of this genetic information to the daughter
Acknowledgments
The authors thank Drs. Kasia Bebenek and Stephanie Nick McElhinny for thoughtful discussion and comments on the manuscript. This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences.
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