Crystal structure of the human Polϵ B-subunit in complex with the C-terminal domain of the catalytic subunit

Abstract

The eukaryotic B-family DNA polymerases include four members: Polα, Polδ, Polϵ, and Polζ, which share common architectural features, such as the exonuclease/polymerase and C-terminal domains (CTDs) of catalytic subunits bound to indispensable B-subunits, which serve as scaffolds that mediate interactions with other components of the replication machinery. Crystal structures for the B-subunits of Polα and Polδ/Polζ have been reported: the former within the primosome and separately with CTD and the latter with the N-terminal domain of the C-subunit. Here we present the crystal structure of the human Polϵ B-subunit (p59) in complex with CTD of the catalytic subunit (p261_C). The structure revealed a well defined electron density for p261_C and the phosphodiesterase and oligonucleotide/oligosaccharide-binding domains of p59. However, electron density was missing for the p59 N-terminal domain and for the linker connecting it to the phosphodiesterase domain. Similar to Polα, p261_C of Polϵ contains a three-helix bundle in the middle and zinc-binding modules on each side. Intersubunit interactions involving 11 hydrogen bonds and numerous hydrophobic contacts account for stable complex formation with a buried surface area of 3094 Ų. Comparative structural analysis of p59–p261_C with the corresponding Polα complex revealed significant differences between the B-subunits and CTDs, as well as their interaction interfaces. The B-subunit of Polδ/Polζ also substantially differs from B-subunits of either Polα or Polϵ. This work provides a structural basis to explain biochemical and genetic data on the importance of B-subunit integrity in replisome function in vivo.

Introduction

In eukaryotes, the bulk of replication is performed by the members of the B-family DNA polymerases Polδ and Polϵ,³ which participate in lagging and leading DNA strand synthesis, respectively (1–3). The other members of this family are Polα and Polζ. Polα extends RNA primers with a short patch of deoxyribonucleotides, and then synthesis is switching to Polδ and Polϵ (4–6). Polζ is one of the key players in replication of damaged DNA (7–9). The common architectural features of all four polymerases are the catalytic subunits with exonuclease/polymerase module(s) and the C-terminal domain (CTD) and the B-subunits (10). Historically, subunits in different organisms were named differently, and the current nomenclature of polymerase subunits is shown in Table 1. The B-subunits stabilize the catalytic subunit (11, 12), act as scaffolding proteins to mediate interactions with other components of the replication machinery (13–16), and regulate DNA-synthesizing activity in a cell cycle-dependent manner, during checkpoints, and telomere maintenance (17–21).

Table 1.

Nomenclature of subunits of B-family DNA polymerases

Polymerase	Subunit	Protein name			Alternative names (both organisms)
		Common	Yeast	Human
Primase-Polα	Small of primasea	PRIM1	Pri1	p49	PriS; p48
	Large of primase	PRIM2	Pri2	p58	PriL
	Aa	POLA1	Pol1	p180	POLA; NSX; Cdc17
	B	POLA2	Pol12	p70	p68
Polϵ	Aa	POLE1	Pol2	p261	POLE; CRCS12; FILS
	B	POLE2	Dpb2	p59	DPE2
	Third	POLE3	Dpb3	p17	CHRAC17
	Fourth	POLE4	Dpb4	p12	CHRAC16; YHHQ1
Polδ	Aa	POLD1	Pol3	p125	Cdc2; MDPL; CRCS10
	B	POLD2	Pol31	p50	Cdc1; HYS2
	C	POLD3	Pol32	p66	Cdc27
	Fourth	POLD4	Cdm1b	p12	POLDS
Polζ	Aa	REV3L	Rev3	p353	POLZ
	B	POLD3	Pol31	p50	Cdc1; HYS2
	C	POLD4	Pol32	p66	Cdc27
	Small	REV7	Rev7	p30	MAD2L2; MAD2B; FANCV; POLZ2

^a Small subunit of primase and A subunits of DNA polymerases contain the catalytic center(s).

^b This gene exists in Schizosaccharomyces pombe, and its homolog is absent in S. cerevisiae.

Human Polϵ consists of four subunits (Table 1): the catalytic subunit (p261) and the auxiliary subunits (the second subunit, p59, also referred to as the B-subunit, and the third and fourth subunits, p17 and p12, respectively) (10). p261 is a bi-lobe protein comprised of two tandem exonuclease/polymerase domains, the first of which is active, whereas the second is inactive and, along with CTD, plays an essential structural role (16, 22–24). CTD interacts with p59 and has eight conservative cysteines coordinating two zinc ions (25, 26). In yeast, deletion of CTD abolishes interaction between the catalytic and B-subunit and is lethal (23, 27). The integrity of the CTD–B-subunit complex is important for stability of the genome (28, 29). Auxiliary subunits play a regulatory role and guide Polϵ to different biological pathways. The B-subunit integrates Polϵ into the eukaryotic replisome, and in yeast, its N-terminal helical domain is essential for assembly of the Cdc45–MCM–GINS helicase (15). The small subunits p17 and p12 interact with each other through histone-fold motifs similar to those found in histones H2A and H2B (30). As a heterodimer, they bind p261 at the second, inactivated exonuclease/polymerase domain and increase Polϵ affinity to double-stranded DNA (31, 32). Small subunits are not essential for cell growth, but their deletion in yeast destabilizes the replication fork and elevates spontaneous mutagenesis (33).

The crystal structure of the human Polδ B-subunit (p50) in complex with the N-terminal domain of the C-subunit (p66_N) provided the first view of the three-dimensional organization of B-subunits (34). The docking surface of Polδ CTD on the B-subunit has been predicted (25) using structural and mutational analyses (34, 35). Moreover, structural and biochemical studies led to a discovery that the CTD of human Polζ is capable of binding the p50–p66 complex of human Polδ in the same manner as the CTD of Polδ (25), meaning that both the Polζ and Polδ catalytic subunits form a complex with p50–p66. These studies led to experiments that confirmed this discovery in yeast (9). The crystal structures of the yeast and human Polα B-subunits in complex with CTDs of the catalytic subunits have also been reported as separate complexes (36, 37), as well as within the human primosome (38). Until now, the structure of the Polϵ B-subunit was the only one unsolved among the human B-family DNA polymerases. Here we report the crystallization, structure determination, and comparative structural analysis of the full-length human Polϵ B-subunit (p59) in complex with the CTD of the catalytic subunit (p261_C).

Results

Overall structure of the human Polϵ CTD–B-subunit complex (p59–p261_C)

The crystal of the p59–p261_C complex contains two independent molecules in an asymmetric unit with root mean square deviations (RMSD) of 0.25 Å for 552 superimposed α-carbons. Each molecule is comprised of p261_C (residues 2142–2286) and full-length p59 (residues 1–527) (Fig. 1A). The density was absent for the N-terminal residues 1–83 and for the loop region 146–162 in both p59 subunits, as well as for the N- and C-terminal tails (2142–2149 and 2174–2182) and loop 2283–2286 in both p261_C domains. The molecule has a slab-like shape with overall dimensions of 79 Å × 75 Å × 53 Å (Fig. 1B).

Figure 1.

Overall structure of Polϵ p59–p261_C. A, schematic representation of the domain organization. The dark blue lines in the schematics of p261_C present the relative positions of the zinc-coordinating residues in two zinc-binding modules: Zn1 (Cys²¹⁵⁸, Cys²¹⁶¹, Cys²¹⁸⁷, and Cys²¹⁹⁰) and Zn2 (Cys²²²¹, Cys²²²⁴, Cys²²³⁶, and Cys²²³⁸). B, stereo view of p59–p261_C. p261_C is colored light pink; the PDE domain (excluding region 84–121) and OB domain are colored cyan and green, respectively. The N-terminal portion of the PDE domain (residues 84–121) is colored red. Zinc atoms are depicted as dark blue spheres.

C-terminal domain of p261

p261_C adopts an elongated asymmetric shape (Fig. 1) with a three-helix bundle (α₁, α₂, and α₃) in the middle and zinc-binding modules (Zn1 and Zn2) on each side, where the highly conserved cysteine residues form the tetrahedral coordination geometries for the zinc ions. Zn1 contains two pairs of antiparallel β-strands: ↑β₁↓β₄ and ↑β₂↓β₃. Two zinc-coordinating cysteines, Cys²¹⁵⁸ and Cys²¹⁶¹, are from strand β₂ and loop β₂-β₃, respectively. The partially disordered loop β₄–α₁ provides the remaining two Zn-coordinating cysteines: Cys²¹⁸⁷ and Cys²¹⁹⁰. The position of Zn1 relative to the three-helix bundle is stabilized by a hydrophobic core formed between helices α₁ and α₂, strands β₁ and β₄, and loop β₄–α₁. The Zn2 module is more compact than Zn1 and contains a small antiparallel β-sheet, ↓β₆↑β₅↓β₇, and two loops: β₅–β₆ with one zinc-coordinating cysteine, Cys²²²⁴; and β₆–β₇ with two zinc-coordinating cysteines: Cys²²³⁶ and Cys²²³⁸; remaining Cys²²²¹ is located on the strand β5. Zn2 is connected to the three-helix bundle via two linkers, α₁–β₅ and β₇–α₂, but has no additional stabilizing interactions with the rest of p261_C.

B-subunit

The p59 subunit is comprised of three domains: an N-terminal domain (NTD), residues 1–75; a phosphodiesterase-like (PDE) domain, residues 84–121 and 245–527; and an oligonucleotide/oligosaccharide-binding (OB) domain, residues 122–244 (Fig. 1). The absence of the density for residues 1–83 in the crystal suggests that the NTD is connected to the rest of the molecule with a flexible linker and lacks the stabilizing interdomain interactions with the PDE, the OB, and p261_C. The NMR structure of the NTD revealed a globular four-helical bundle that resembles the C domain of AAA+ proteins (39). Because the residues 1–75 are well folded in solution, the flexible linker connecting the NTD with the rest of p59 should be within residues ⁷⁶QSVDETIE⁸³ (Fig. 2). The PDE domain is comprised of a central two-layer β-sheet (layer 1, ↑β₁₉↓β₂₀↓β₁₈↓β₁₇↑β₁₆↓β₃, and layer 2, ↓β₂₂↑β₂₁↓β₁₂↓β₁₃↓β₁₄↓β₁₅). Helices α₇, α₁₃, α₁₄, and 3_10–2 are flanking layer 1, helices α₉–α₁₂ are flanking layer 2, and helix α₈ is located on the side of both layers. The PDE is inactive because it lacks the conserved histidine and aspartate residues involved in metal coordination and catalysis. The OB domain contains a β-barrel (↑β₆↓β₇↑β₈β₉↑β₁₁↓β₁₀), a helix α₅ on the side, and a helix α₆ on the bottom of the barrel. The majority of residues in the loop linking helices α₅ and α₆ are disordered. One of the strands in the barrel is divided by an inserted loop into two strands: β₈ and β₉. Contrary to the NTD, the PDE and OB domains are well stabilized relative to each other. The interaction between them is dominated by polar contacts with 22 well defined interdomain hydrogen bonds.

Figure 2.

Model of the p59–p261_C structure. The model comprised of the crystal structure of p59–p261_C and the NMR structure of NTD (Protein Data Bank code 2v6z) (39). The dashed line indicates the linker between NTD and PDE. The domains are colored as in Fig. 1.

Interaction between p59 and p261_C

The p59–p261_C interface has an elongated shape with complementary surfaces that bury the surface area of 3094 Ų. The subunits are tightly associated by 11 intersubunit hydrogen bonds and by extended hydrophobic areas (Figs. 3 and 4). The N-terminal part of the helix α₂ and the entire helices α₁ and α₃ of the three-helix bundle of p261_C interact mainly with the loops of 3_10–1–α₅, β₁₄–α₁₂, and α₁₄–3_10–2 of the p59 PDE domain. The most prominent feature of this interaction is the wedging of the p59 Phe¹²⁰ side chain deep into the hydrophobic core of the p261_C three-helix bundle (Fig. 4). In addition, the β-sheet and the loop β₆-β₇ of p261_C Zn2 are engaged in interactions with the OB domain of p59, where the p261_C Met²²³² side chain is inserted into the hydrophobic depression between the helix α₅ and the β-barrel of the OB domain. The modular architecture of p261_C suggests the possibility of independent folding and p59 binding for Zn2 and the three-helix bundle. This structural feature may explain the effect of small deletions in the yeast ortholog of p261_C on its affinity to the B-subunit. It was demonstrated by a two-hybrid assay that partial disruption of either Zn2 or the α-helices weakened intersubunit interaction but did not completely abolish it (27). Only the combined deletion of Zn2 and helices α2 and α3 completely eradicated the association between the catalytic and B-subunits; probably, in this case, the folding of the helix α1, as well as its interaction with the B-subunit were affected in the absence of helices α2 and α3. Unlike Zn2, the Zn1 module does not participate in intersubunit interaction, but it is important for the folding of a three-helix bundle, which explains the defect in cell growth upon partial deletion of Zn1 in CTD of yeast Polϵ (23, 27). In contrast to the Zn2 module and the helical bundle of CTD, the OB and PDE domains of the B-subunit are tightly associated, so a defect in either of these domains will affect the integrity of the other one and should completely abrogate the intersubunit interaction. Consistently, all truncation variants of Dpb2 were unable to interact with the catalytic subunit (40).

Figure 3.

Close-up stereo view of the hydrophilic contacts between p261_C and p59. The secondary structure elements are shown with 60% transparency. The domains and carbons are colored according to Fig. 1. The side chains or main chains of the residues involved in intersubunit interactions are shown as sticks. The nitrogens, oxygens, and sulfurs are colored blue, red, and yellow, respectively.

Figure 4.

Close-up stereo view of the hydrophobic contacts between p261_C and p59. The presentation details are described in the Fig. 3 legend.

Discussion

Comparison of the B-subunits of Polα, Polδ, Polϵ, and Polζ

The crystal structures of B-subunit complexes from all human B-family DNA polymerases, including CTDs of catalytic subunits with B-subunits (p70–p180_C of Polα and p59–p261_C of Polϵ) and the B-subunit with a part of the C-subunit (p50–p66_N of Polδ or Polζ), are available now for comparison (Fig. 5).

Figure 5.

Side-by-side comparison of the Polϵ p59–p261_C, Polα p70–p180_C, and Polδ/Polζ p50–p66_N crystal structures. Red arrows indicate the structural elements contributing to significant differences between the three structures. The coloring of molecules is similar to Fig. 1, except the p66_N domain of Polδ/Polζ C-subunit is colored purple. Two different orientations are shown for each molecule. The molecules are displayed using Protein Data Bank entries 5vbn, 5exr, and 3e0j.

The common features of the B-subunits are the presence of a PDE domain with a two-layer β-sheet and an OB domain with a β-barrel, the relative positioning of these two domains, and the surface for docking of the CTD (34, 36, 37). In addition, B-subunits contain some equivalents of helices α₃, α₄, α₅, α₉, and α₁₀ (p59 secondary structure nomenclature is used in all comparisons). Despite the conservation of core elements, the B-subunits exhibit significant shifts in the positions of helices and the structures of loops. As a result, the RMSD between p59 and p70 is 2.9 Ų for 352 matching residues of 427. The similarity between p50 and either p59 (2.9 Ų for 329 residues) or p70 (3.1 Ų for 341 residues) is low as well. Among the notable differences between p59 and p70 or p50 are the topologies of residues N- and C-terminal to the OB domain. The β-hairpin β₂β₃ and helix 3_10–1 from residues N-terminal to the OB domain, together with helix α₇ and coil α₇-α₈ from residues C-terminal to the OB domain, form a bulky addition on the side of p59 (Fig. 5). This provides p59 a relatively globular shape compared with p70. The bulky addition of p59 is more prominent compared with p50, which also lacks the adjacent helix α₈ (Fig. 5). The unique features of p50 are the interaction with p66_N and the absence of a globular NTD linked with a PDE domain via a flexible linker in p59 and p70. The docking of p66_N is performed mainly by p50 helices α9, α10, an α-helix that is inserted into the loop α₉-β₁₃, and the N-terminal residues that are folded as an α-helix, an extended coil, and a small β-strand. In addition, p50 has an elongated strand β₂₂ that interacts with a β-sheet of p66_N, resulting in a continuous nine-strand β-sheet (Fig. 5).

Comparison of CTDs

CTDs have significant differences in their size, structure, and sequence. For example, p125_C of Polδ and p353_C of Polζ are 107 and 100 amino acids (aa) long, respectively, and significantly shorter than p180_C (195 aa) of Polα and p261_C of Polϵ (144 aa). Both p261_C and p180_C contain a three-helix bundle with a Zn1 and a Zn2 on either side (Fig. 5). The aa identity between p125_C and p353_C is 18%, whereas the sequence identities for all other pairs are marginal and comparable with randomly selected sequences. Secondary structure predictions for p125_C and p353_C show that both contain a short (10-aa) α-helix N-terminal to Zn1 and two longer α helices (28 and 22 aa) (Fig. 6B) that supposedly fulfill the role of α₁ and α₂ in p261_C and p180_C (Fig. 6A). In contrast to p180 and p261, p125 and p353 coordinate a [4Fe-4S] cluster at the second metal-binding site of their CTDs (25, 41). Interestingly, two of the [4Fe-4S] coordinating cysteines are predicted to be from the C-terminal portion of helix α₂, and both the Zn1 and [4Fe-4S]-binding modules contain short loops (10 aa or less) and no β-strands. Therefore, they acquire a compact structure that is different from the corresponding Zn1 and Zn2 modules of p180_C and p261_C. At present, only the latter two domains are available for detailed comparison. Their superimposition indicates the conservation of helix α₁ and significant differences in the length, conformation, and relative positions of helices α₂ and α₃ (Fig. 7). The prominent feature of p180_C is a longer helix α3 and two helices inserted between the helices α₂ and α₃ that stabilize an elongated loop in the Zn1. The corresponding loop is disordered in p261_C. Additionally, p180_C contains a C-terminal extension that forms a β-strand and an α-helix upon interaction with the large subunit of primase (38, 42).

Figure 6.

Structure-based sequence alignments of p261_C with p180_C (A) and p125_C with p353_C (B). Blue- and green-shaded boxes depict the α-helices and β-strands, respectively. The gray-shaded boxes indicate the residues that are disordered in the crystal structures of p261_C and p180_C. The secondary structure elements are from the crystal structures of p261_C and p180_C (A) and from Phyre (57) predictions for p125_C and p353_C (B). The conserved residues are highlighted in red. Magenta-shaded boxes depict the zinc-coordinating cysteines. Yellow bars indicate the sequences comprising the zinc-binding modules Zn1 and Zn2. The red bar indicates the sequences implicated in iron–sulfur cluster binding.

Figure 7.

Differences between the Polϵ p261_C and Polα p180_C structures. The molecules were aligned by superposition of the conservative helices α₁ and α₂. p261_C and p180_C are colored light pink and gray, respectively.

Structural insight into the Polϵ p59–p261_C subcomplex confirms our previous observation that the Zn2 module of human Polϵ requires zinc for specific docking on p59 (25), which is important for the formation of a stable complex between the catalytic and the B-subunit. It is shown in Ref. 25 that p261_C expressed and purified alone contains a substantial level of iron, whereas the pure and stoichiometric p59–p261_C complex is iron-free. This indicates that the Zn2 module of p261_C is able to coordinate the iron–sulfur cluster, but this prevents specific interaction with p59. According to the structure of [4Fe-4S] cluster-containing proteins, for example, human primase (43), coordination of the cluster by p261_C would require the distances between zinc-binding cysteines to be increased by ∼3 Å, which would definitely change the Zn2 structure and result in steric hindrance with the OB domain. These peculiarities explain why an iron–sulfur cluster was seen in partially purified samples of yeast Polϵ and its CTD (41). In contrast to the other B-family DNA polymerases, an additional zinc-binding module is located in the catalytic domain of Polϵ (44), and the iron–sulfur cluster can be misincorporated at this position as well (45). Thus, all structural data support our previous conclusion (25) that Polϵ and Polα require only zinc for correct assembly (5, 44). Moreover, concentrated and very pure samples of human Polα and Polϵ, obtained in our laboratory, were iron-free and fully functional (46, 47), indicating that their operation in eukaryotic cells is not dependent on the iron–sulfur cluster assembly machinery.

Interactions between CTD and the B-subunit

The structures of Polα p70–p180_C (36) and Polϵ p59–p261_C (Fig. 1B) revealed the common features as well as significant differences in the mode of intersubunit interactions. In both molecules, Zn2 mainly interacts with the OB domain, whereas the helical bundle of CTD associates with the PDE domain of the B-subunit. In both cases, the contact area between Zn2 and the B-subunit composes ∼30% of the total interaction interface between the two subunits. With regard to the differences, it is worth noting the predominance of hydrophobic contacts at the CTD–B-subunit interaction interface and the reduction of the buried surface area by 30% in the case of Polϵ (Figs. 3 and 4). Superimposition of the B-subunits in p70–p180_C and p59–p261_C shows a significant shift in position of p261_C relative to p180_C with an average displacement of their central three-helix bundles exceeding 6 Å (Fig. 8).

Figure 8.

Comparison of CTD docking on the B-subunit between p59–p261_C and p70–p180_C. Both CTD–B-subunit complexes were aligned by superposition of the B-subunits; p70 is omitted for clarity. The p59–p261_C domains are colored according to Fig. 1, and p180_C is colored gray.

The structure of p50 with either p125_C of Polδ or p353_C of Polζ has not been reported yet. The mapping of the p50 surface area responsible for the docking of p125_C was performed with data obtained for the fission yeast ortholog using random and directed mutagenesis techniques (35). This revealed that the p50 residues responsible for p125_C interaction are disordered. It was proposed and confirmed that disordered loops provide the ability of p50 to adopt the binding of both p125_C and p353_C despite their low amino acid sequence identity (25). Unlike p50, p70 of Polα and p59 of Polϵ accept only one CTD: p180_C or p261_C, respectively.

The p59–p261_C structure helps to explain phenotypes of the yeast Dpb2 mutants

A set of temperature-sensitive Dpb2 alleles was previously generated by using direct and random mutagenesis techniques (Table 2). Some Dpb2 mutants exhibited a mutator phenotype comparable to that of mismatch-repair-defective strains (msh6), indicating that defects in the B-subunit of the human Polϵ gene can induce genomic instability. The structure of p59–p261_C provides a rationale for explanation of the effects of amino acid changes in the yeast Polϵ B-subunit, characterized by weakened interaction with the catalytic subunit and the GINS complex, elevated spontaneous mutagenesis, and the affected Nrm1 branch of the DNA replication checkpoint (11, 21, 28, 29, 40, 48).

Table 2.

Correlation between predicted effect on protein integrity and phenotypes of Dpb2 mutants

The data for cell growth, mutability, and interaction between the catalytic and B-subunits of yeast Polϵ were obtained from Refs. 11, 28, and 40. Amino acid changes resulting in steric hindrance are indicated by an asterisk. Mutations resulting in hydrophobic pocket disturbance caused by hydrophobic amino acid replacement by a charged or significantly smaller residue are shown in bold type. p59 residues near the intersubunit interaction interface (when the distance to the closest p261_C residue is <10 Å) are underlined; p59 resides at the interaction interface are underlined and italicized. It was assumed that mutations resulting in steric hindrance or affecting a hydrophobic pocket have a moderate effect on B subunit integrity; otherwise, they have a minor effect.

Dpb2 allelea	Amino acid substitutions in Dpb2 (homologous amino acid of p59 is in parenthesis)	Cell growth at 37 °Cb	Pol2 bindingc	Mutability increase at CanRd	Integrity losse
			%
1	D300 (D199)N, K521 (P394)R, V565 (L435)F*, G662 (G500)R*	+f	17	(2.4)g	++
100	L284 (V183)P*, T345 (G241)A	−	0.6	7 (5.4)	++
101	L284 (V183)P*	±	83	4 (2.6)	++
102	T345 (G241)A	+	106	(1.4)	+
103	T342 (N238)I, S343 (A239)F, T345 (G241)I, P347 (P243)S, P348 (P244)S	−	53	5 (5.2)	++
104	I484 (V364)S, L630 (L469)S, F649 (F487)C	−	15	3	++
107	K171 (N87)E, S182 (V98)P*, L284 (V183)P*, I385 (K276)T, M572 (L435)V, S574 (A436)T	−	5	7	++
108	T269 (K166)A, H318 (L217)R*, Y320 (Y218)S, I359 (Y255)V, T629 (A468)A, I635 (V474)V	−	47	2	++
109	K197 (M111)M, N258 (H155)S, S453 (N333)C, C520 (V393)L*, F615 (L454)S	±	27	2	++
110	L285 (L184)W*, L365 (F261)S, N405 (D292)I*, M572 (L435)K	−	3	4	++
111	Y223 (T135)H	+	103	1.3	+
112	E598 (I437)D, P621 (P460)S, L641 (I480)H*, I665 (P503)V, T682 (V519)A	−	2	7	++
113	K171 (N87)E, S182 (V98)P*, L284 (V183)P*	−	0.7	8	++

^a Alleles dpb2-105 and dpb2-106 containing more than 11 mutations are not listed.

^b Normal and slow cell growth at elevated temperature is designated as + and ±, respectively; − indicates very slow or no cell growth.

^c Binding experiments were conducted using the yeast two-hybrid system; β-galactosidase activity measured at 23 °C was taken for 100% in the case of a wild-type protein.

^d The frequency of forward mutations was measured at the CAN1 locus (the numbers indicate the fold increase compared with a wild-type protein); the DPB2 variants were integrated into the DPB2 chromosomal locus (ΔI(-2)I-7BYUNI300).

^e Integrity loss is a degree of fold disturbance defined as minor (+) or moderate (++).

^f In one study, cells demonstrated normal growth at 37 °C when the allele dpb2-1 was located on a centromeric plasmid, but the authors were unable to obtain viable integrants of this allele on the chromosome (28). In an earlier study, cells with a chromosomal copy of dpb2-1 stopped division within 4 h after a temperature shift up to 38 °C and exhibited slow growth even at 24 °C (11).

^g The values in parentheses are obtained from experiments where DPB2 variants are located on a centromeric plasmid.

High secondary structure conservation between p59–p261_C and its yeast ortholog (Fig. 9) implies similar three-dimensional organization, allowing for mapping the residues changed in Dpb2 of Saccharomyces cerevisiae on the structure of p59 and for predicting the effect of these mutations on the integrity of Dpb2 and its interaction with the catalytic subunit (Table 2). Structural analysis of 38 mutations revealed that two residues, Ser⁴⁵³ and Pro⁶²¹, are located at the CTD–B-subunit interface. According to modeling, Asn³³³ of p59, which corresponds to Ser⁴⁵³ of Dpb2, is located at the edge of the intersubunit interface and does not participate in the interaction with p261_C (Fig. 10A); there is also low probability that the hydroxyl of Dpb2 Ser⁴⁵³ makes a hydrogen bond with any surrounding residue. It is likely that the appearance of a cysteine at this position does not affect the B-subunit folding in yeast and human Polϵ but can result in moderate steric hindrance with Val²¹⁴⁰ of Pol2_C, which corresponds to Ser²¹⁹⁷ of p261_C (Fig. 9B). In the case of the Dpb2-112 mutant, Pro⁴⁶⁰ of p59, which corresponds to Pro⁶²¹ of Dpb2, participates in hydrophobic interactions with three amino acids of p261_C: Met²²¹², Thr²²¹⁵, and Leu²²¹⁶ (Fig. 10B), which correspond to Glu²¹⁵⁵, Leu²¹⁵⁸, and Ile²¹⁵⁹ of Pol2_C, respectively (Fig. 9B). Therefore, the replacement of proline by serine at this position affects the hydrophobic pocket at the CTD–B-subunit interface. Pro⁴⁶⁰ also plays a structural role for the coil α₁₄–3_10–2, and its substitution may affect the position of amino acids Leu⁴⁵⁶, Tyr⁴⁵⁷, Tyr⁴⁶², and Trp⁴⁶³, which interact with p261_C (Fig. 9A). Thus, P621S substitution should have stronger effect on intersubunit interaction than S453C, which is well correlated with more pronounced effects on viability, affinity to Pol2, and spontaneous mutagenesis in cells expressing Dpb2-112 mutant versus Dpb2-109 (Table 2). Interestingly, several other Dpb2 mutants do not have amino acid substitutions, which can directly affect the association with a catalytic subunit, but some of them have a phenotype similar to the Dpb2-112 mutant (Table 2). It is likely that Dpb2 folding is affected in these mutants. All temperature-sensitive mutants listed in Table 2 have a minor to moderate effect on B-subunit integrity. Two alleles with predicted minor effect on Dpb2 folding (dpb2-102 and dpb2-111) have phenotypes close to the wild type.

Figure 9.

Structure-based sequence alignments of the B-subunits (A) and CTDs (B) of human and yeast Polϵ. The α-helices and β-strands are depicted by green- and blue-shaded boxes, respectively. The gray-shaded boxes indicate the residues that are disordered in the crystal structure of p59–p261_C. The conserved residues are shown in red. The blue, pink, and black circles indicate amino acids involved in intersubunit hydrophobic, hydrophilic, and both types of interactions, respectively. The Dpb2 residues listed in Table 2 are underlined. The fold prediction for the yeast Polϵ subunits was performed using Phyre (57).

Figure 10.

Modeling of the amino acid changes mapped to the intersubunit interface. A, modeling of p59 Cys³³³ and p261_C Val²¹⁹⁷ in place of asparagine and serine, respectively. Only one conformation of Cys³³³ is allowed to avoid steric hindrance with main-chain atoms. In both allowed conformations of Val²¹⁹⁷ (only one is shown for clarity), there is a clash with Cys³³³. B, modeling of p59 Ser⁴⁶⁰ in place of proline. The p59–p261_C domains are colored according to Fig. 1. Carbons of the modeled residues are colored purple. A blue dashed line depicts the distance between atoms. Zn1 and Zn2 labels show the position of corresponding zinc-binding modules.

We propose that mutations affecting hydrophobic pockets or resulting in steric hindrance can lead to local disturbance of the protein fold, and the effect on association of subunits should be stronger as the distance between the mutated residue and the interaction interface becomes shorter. The exact effect of amino acid substitution on the Dpb2 structure depends on many factors, such as location, involvement in interactions with other residues, degree of steric hindrance, and how the molecule can tolerate this change. The contribution of some of these factors, as well as the cumulative effect of several mutations on Dpb2 integrity, is difficult to estimate accurately, especially from the structure of a homologous but diverged protein. Despite these complications, it was possible to make unambiguous structural predictions for several Dbp2 mutants. For example, the Dpb2-113 mutant has a strongest mutator effect coinciding with disrupted binding to the catalytic subunit (Table 2). This mutant has substitution of two residues located on β4 and β6 strands to proline, which results in steric hindrance with a main-chain oxygen of an amino acid located on the neighbor strand. For example, Val¹⁸³ of p59, corresponding to Leu²⁸⁴ of Dpb2, is located in the hydrophobic pocket inside of the β-barrel of the OB domain (Fig. 11A). Steric hindrance between proline in this position and Ala²²⁶ will result in disruption of at least two hydrogen bonds and local disturbance of the β-barrel nearby the CTD–B-subunit interface. These two proline substitutions in the Dpb2-113 mutant have an additive effect, because the Dpb2-101 mutant with only one proline substitution shows mild phenotype (Table 2). It is likely that only these two proline substitutions define the phenotype of the Dpb2-107 mutant, which has four additional mutations with predicted minor effects on protein integrity. The other allele with a severe defect in intersubunit association is a dpb2-110, which has three mutations with a predicted moderate effect on the protein fold. Probably, the L285W change is the main determinant of the phenotype of this allele. The conservative Leu²⁸⁵ is located inside of a hydrophobic pocket between the α-helix and the β-barrel of the OB domain, close to the intersubunit interface. Modeling of a bulky tryptophan at this position reveals strong steric hindrance with surrounding hydrophobic residues, which will disturb the OB domain, as well as its interaction with the PDE domain and CTD (Fig. 11B).

Figure 11.

Modeling of the amino acid changes inducing steric hindrance. A, close-up view of a hydrophobic pocket with modeled proline in place of Val¹⁸³. Val¹⁸³ makes two main-chain–to–main-chain hydrogen bonds with Ala²²⁶ (not shown). Leu¹⁷¹, Val¹⁸³, Leu¹⁹⁷, Ala²²⁶, and Phe²³⁵ of p59 correspond to Leu²⁷⁴, Leu²⁸⁴, Leu²⁹⁸, Val³²⁸, and Phe³³⁹ of Dpb2, respectively. B, close-up view of a hydrophobic pocket with modeled tryptophan in place of Leu¹⁸⁴. In the shown conformation, tryptophan clashes with the side chains of Pro²⁴³ and Leu¹³⁷. Tyr¹³⁴, Leu¹³⁷, Thr¹⁴¹, Leu¹⁶⁵, Leu¹⁸⁴, Phe²²³, Leu²²⁵, and Pro²⁴³ of p59 correspond to Tyr²²², Thr²²⁵, Val²²⁹, Ile²⁶⁸, Leu²⁸⁵, Met³²⁵, Leu³²⁷, and Pro³⁴⁷ of Dpb2, respectively. p59 and p261_C are colored according to Fig. 1. Carbons of the modeled residues are colored purple. The residues are shown as sticks. The secondary structure elements are shown with 20% transparency. Blue dashed lines depict the closest distance between the residues. Proline and tryptophan rotamers, shown in A and B, respectively, correspond to minimal steric hindrance.

The most interesting example of the influence of B-subunit integrity on the replisome function in vivo is a dpb2-200 mutation, which results in lethality at all temperatures and corresponds to deletion of the last six residues (QEEIYI) from the C terminus of the yeast Polϵ B-subunit and to substitution of conservative Pro⁶⁷⁷ by serine (49). Mapping of these mutations on the p59 structure led to the conclusion that this change induces severe destabilization of the protein fold (Figs. 9A and 12). The C-terminal deletion disrupts the last β-strand, which is a part of a central two-layer β-sheet of the PDE domain. Moreover, Pro⁵¹⁴ and Leu⁵²⁴ of p59, which correspond to Pro⁶⁷⁷ and Ile⁶⁸⁷ of Dpb2, participate in the formation of two hydrophobic pockets tethering different parts of the PDE domain (Fig. 12).

Figure 12.

Analysis of the role of Pro⁵¹⁴ and the extreme C-terminal region on p59 folding. A, close-up view of a hydrophobic pocket containing Leu⁵²⁴ and Phe⁵²⁷. The carbons of p59 residues corresponding to the deleted region in the Dpb2-200 mutant are colored purple. Lys³⁰³, Ile³⁰⁶, Met³⁰⁷, Phe⁵⁰², Phe⁵⁰⁷, and Phe⁵⁰⁹ of p59 correspond to Ala⁴¹⁶, Lys⁴¹⁹, Ile⁴²⁰, Phe⁶⁶⁴, Ala⁶⁷⁰, and Tyr⁶⁷² of Dpb2, respectively. B, close-up view of a hydrophobic pocket containing Pro⁵¹⁴. Ala²⁸⁵, Phe²⁸⁷, Tyr⁴⁰⁷, and Phe⁵¹² of p59 correspond to His³⁹⁷, Phe³⁹⁹, Tyr⁵³⁴, and Tyr⁶⁷⁵ of Dpb2, respectively. The p59 domains are colored according to Fig. 1. Side chains of the residues are shown as sticks.

These observations support the idea that the effect of Dpb2 mutations on genomic stability is due to disturbance of replication initiation and elongation because of affected polymerase integrity and tethering to the replisome (29). Indeed, mutations of Psf1, which is a component of the GINS complex and interacts with NTD of the Polϵ B-subunit, also increase spontaneous mutagenesis (48). Probably, in Dbp2 mutants with compromised binding to the catalytic subunit, NTD is folded correctly and supports CMG assembling (15) if solubility of the B-subunit and its transport to the nucleus are not significantly affected.

Conclusions

The B-subunits of B-family DNA polymerases have evolved as the hubs for a variety of regulatory protein-protein interactions. The analysis of their crystal structures helps to evaluate the possible mechanisms for the involvement of such hubs during various stages of DNA replication. Recent cryo-EM structure of the yeast CMG-Polϵ-Ctf4-Polα complex indicates that Polϵ interacts directly with the CMG helicase, whereas Polα is located at a distance from the CMG and has more mobility (50). Contrary to the short NTD-PDE linker in human Polϵ, the corresponding linker in human and yeast Polα can be extended to >200 Å, enabling the primosome to reach the priming sites on both the lagging strand and distantly located leading strand (51). Although the role of p70 NTD in Polα tethering to the replisome has not been ascertained thus far, the potential replisome-anchoring site on the N terminus of the catalytic subunit is connected with the rest of the molecule by a 190-aa linker (5). The NTD-PDE linker in Dpb2 is longer than in p59, but it might be structured and compact, because secondary structure prediction shows the presence of two α-helices (Fig. 9A).

The crystal structure of p59–p261_C as provided here is the first high-resolution three-dimensional view of the Polϵ CTD–B-subunit complex. Together with the structure of the polymerase/exonuclease domain of a yeast ortholog (44), this is the only high-resolution structural information for eukaryotic Polϵ available to date. The structures of small subunits (p17, p12) and a catalytically inactive polymerase domain between the catalytic core and p261_C are required to build an accurate model of the whole four-subunit complex including the data from electron microscopy studies of Polϵ or its complex with CMG. Unfortunately, the current resolution of electron microscopy data for Polϵ is not sufficient for unambiguous docking of the better-resolved crystal structures of its parts (16, 24, 50). Further research will be required to evaluate the function of p59 and to find the exact position of p59–p261_C relative to the catalytic core and other domains of Polϵ in the replicating complex. It will also be interesting to see the detailed mechanism of p59–p261_C participation in recruitment of Polϵ to the CMG helicase.

Experimental procedures

Protein expression and purification

Cloning, expression, and purification to homogeneity of the full-length human Polϵ B-subunit (p59; residues 1–527) in complex with the C-terminal domain of the catalytic subunit (p261_C; residues 2142–2286) have been described elsewhere (25). The peak fractions were combined and concentrated to 11 mg ml⁻¹ in buffer containing 10 mm Tris-HCl, pH 7.7, and 1 mm DTT. The concentrated protein solution was frozen in liquid nitrogen and stored at 193 K until use.

Crystallization

The aliquots of protein solution were defrosted and centrifuged, and the absence of aggregation was verified with dynamic light scattering. Initial crystallization screening was performed by the sitting-drop vapor diffusion method at 295 K by mixing 1 μl of protein solution with 1 μl of reservoir solution. The reservoirs contained 2-fold diluted Crystal Screen and Crystal Screen 2 (Hampton Research) solutions with the addition of 2 mm Tris(2-carboxyethyl)phosphine, pH 7.5. Clusters of crystals appeared in the 32nd condition of Crystal Screen. The optimized reservoir solution with 50 mm sodium citrate, pH 5.6, 0.70 m ammonium sulfate, and 2 mm Tris(2-carboxyethyl)phosphine resulted in well shaped single crystals (Fig. 13) growing at 295 K in 3–5 days.

Figure 13.

Photomicrograph of the p59–p261_C crystal.

Data collection

For diffraction data collection, the crystals were soaked in a cryoprotectant solution (50 mm sodium citrate, pH 5.6, 1 m ammonium sulfate, and 32% (v/v) glycerol) for a few seconds, scooped in a nylon-fiber loop, and flash-cooled in a dry nitrogen stream at 100 K. Diffraction data sets were collected using synchrotron X-rays on the Argonne National Laboratory Advanced Photon source beamline 24ID-C equipped with a Pilatus 6M detector. All intensity data were indexed, integrated, and scaled with DENZO and SCALEPACK from the HKL-2000 program package (52). The crystals belong to tetragonal space group P2₁2₁2 and diffract up to 2.35 Å resolution. The crystal parameters and data processing statistics of the best data set are summarized in Table 3.

Table 3.

Summary of data collection and refinement statistics

Data collection
Space group	P21212
Cell dimensions (Å)
a, b, c	92.26, 201.46, 78.34
Resolution (Å)	40–2.35 (2.39–2.35)a
Rmerge	0.072 (0.481)
I/σI	21.5 (2.2)
Completeness (%)	98.6 (98.2)
Unique reflections	61,804 (3044)
Redundancy	3.3 (3.1)
Refinement
Resolution (Å)	39.75–2.35 (2.5–2.35)
No. reflections	58,410 (8677)
Rwork/Rfree	0.225/0.264 (0.329/0.374)
No. atoms
Protein	8829
Ligand/ion	14
Water	212
Mean B-factors (Å2)	46.0
R. M. S. deviations
Bond lengths (Å)	0.009
Bond angles (°)	1.5
Ramachandran plot (%)
Core	87.7
Allowed	11.0
Generously allowed	1.3
Disallowed	0

^a The numbers in parentheses refer to the highest-resolution shell.

Crystal structure determination

The crystal structure of the p59–p261_C complex was determined by the single-wavelength anomalous dispersion method. The positions of four zinc ions were located from anomalous difference Patterson maps, indicating the presence of two independent p59–p261_C molecules in asymmetric unit. Initial automated model building performed with Phenix (53) revealed about 65% of correctly built structure. The rest of the crystallographic computing was performed with CNS version 1.1 (54) using phases from the automatically built partial model. Non-crystallographic symmetry restraints and density modification with 2-fold averaging were applied during the initial stages of model refinement and map calculations for manual model building with Turbo-Frodo. Application of zonal scaling (55) slightly improved the quality of the electron density maps. After the addition of solvent molecules, the model was refined to an R_cryst of 22.2% and an R_free of 26.28% at the highest resolution of 2.35 Å. The final refinement statistics are provided in Table 3. The figures containing molecular structures were prepared using the PyMOL Molecular Graphics System (version 1.8; Schrödinger) (56); amino acids were modified using the mutagenesis tool and the library of backbone-dependent rotamers included in this software.

Author contributions

A. G. B. and J. G. expressed and purified the p59–p261C complex. N. D. B. and J. G. crystallized the complex. I. K., N. D. B., and T. H. T. collected the diffraction data. Y. I. P. analyzed genetic data. T. H. T. initiated the project and determined the crystal structure. T. H. T. and A. G. B. wrote the manuscript, with contributions and critical comments from the other authors.