Human Pol Ζ Purified with Accessory Subunits Is Active in Translesion DNA Synthesis and Complements Pol Η in Cisplatin Bypass

Human Pol ζ purified with accessory subunits is active in translesion DNA synthesis and complements Pol η in cisplatin bypass

Young-Sam Lee1, Mark T. Gregory, and Wei Yang2

Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892

Contributed by Wei Yang, December 27, 2013 (sent for review December 5, 2013) DNA polymerase ζ (Pol ζ) is a eukaryotic B-family DNA polymerase a nucleotide opposite either a cis–syn thymine or a 6-4 photo- that specializes in translesion synthesis and is essential for normal product (23). Genetic data indicate that a complete lesion bypass embryogenesis. At a minimum, Pol ζ consists of a catalytic subunit event may require two TLS DNA polymerases (24)—one for Rev3 and an accessory subunit Rev7. Mammalian Rev3 contains nucleotide incorporation opposite a lesion (insertion step) and >3,000 residues and is twice as large as the yeast homolog. To the other for the subsequent primer extension (extension step). date, no vertebrate Pol ζ has been purified for biochemical char- The insertion step of TLS is often accomplished by a Y-family acterization. Here we report purification of a series of human Rev3 polymerase, whose active site is uncommonly large, solvent- deletion constructs expressed in HEK293 cells and identification of exposed, and flexible (25). Studies of another B-family TLS DNA a minimally catalytically active human Pol ζ variant. With a tagged polymerase from Escherichia coli (Pol II) show that it efficiently form of an active Pol ζ variant, we isolated two additional acces- extends a DNA primer after a lesion by looping out the damaged sory subunits of human Pol ζ, PolD2 and PolD3. The purified four- DNA template strand, leading to frameshift and mixed-type subunit Pol ζ4 (Rev3–Rev7–PolD2–PolD3) is much more efficient mutations (26). and more processive at bypassing a 1,2-intrastrand d(GpG)-cisplatin In budding yeast, REV3 has been shown to be epistatic with cross-link than the two-subunit Pol ζ2 (Rev3–Rev7). We show that POL32, a subunit of DNA Pol δ. Inactivating either REV3 or η POL32 – complete bypass of cisplatin lesions requires Pol to insert dCTP leads to reduced spontaneous mutagenesis (27 29). As BIOCHEMISTRY opposite the 3′ guanine and Pol ζ4 to extend the primers. with all eukaryotic B-family DNA polymerases, Rev3 contains a Cys-rich C-terminal domain (CTD) (30–33), which forms a TLS | REV3L | MAD2L2 | processivity | two-polymerase lesion bypass zinc-finger domain followed by a [4Fe–4S] cluster (34). In Pol α, δ, and e, each CTD interacts with its specific accessary subunits NA polymerase ζ (Pol ζ), composed of the catalytic Rev3 (32, 35). Recently, three groups have independently shown that Dand accessary Rev7 subunits, is an error-prone DNA the [4Fe–4S] cluster of yeast Rev3 interacts with Pol31 and Pol32 translesion polymerase that causes both spontaneous and DNA subunit (36), thus forming an stoichiometric four-subunit Pol ζ damage-induced mutagenesis (1, 2). More than two-thirds of the (Pol ζ4; Rev3–Rev7–Pol31–Pol32) (23, 37, 38). Baranovskiy et al. 1,504 residues in yeast Rev3 share sequence homology with all further showed that the CTDs of human Pol ζ and δ share the same B-family DNA polymerases, including Pols α, δ, and e, which are accessary subunits p50 and p66, homologs of yeast Pol31 and responsible for the bulk of high-fidelity genomic replication in Pol32, respectively (37). The interaction between yeast Rev3 eukaryotes (3). Unlike the typical B-family polymerases, Pol ζ and Pol31 is reported to be direct, and Pol32 is essential to lacks an intrinsic 3′–5′ exonuclease activity and thus has no stabilize Pol31 and, furthermore, via its interactions with pro- proofreading function (2). Human homologs of REV3 (REV3L) liferating cell nuclear antigen (PCNA), recruits and activates Pol ζ and REV7 (MAD2L2; hereafter referred to as REV7) genes were to carry out TLS (38). The catalytic activity of yeast Pol ζ is im- identified shortly after yeast Pol ζ was characterized. Human proved by the presence of Pol31 and Pol32 (23, 38). Rev3 contains 3,130 residues and is twice as large as the yeast counterpart (4). Human and yeast Rev7 are homologous (5) and Significance bear sequence similarity to the mitotic checkpoint proteins Mad2 Saccharomyces cerevisiae REV3 REV7 (6). Unlike and genes, Although human DNA polymerase ζ (Pol ζ) is essential for DNA which are nonessential and whose knockout leads only to a de- Rev3l replication and cell proliferation, difficulties purifying active creased rate of damage-induced mutagenesis (7, 8), knock- Pol ζ have hindered its biochemical characterization. We report Rev3l−/− out in mice is embryonic-lethal (9), and mouse embryonic here the first purification of an active form of human Pol ζ stem cells are not viable (10, 11). Human and mouse cell cultures Rev3l holoenzyme composed of Rev3, Rev7, PolD2, and PolD3, which obtained from conditional knockout show genome instability opens up the possibility for detailed biochemical and structural and growth defects without an external challenge of DNA damage studies of this essential enzyme. Based on genetic data, it has – ζ (12 14). DNA pol is apparently essential for normal cell pro- been postulated that two specialized DNA polymerases are liferation and embryogenesis in mammals. needed for successful translesion synthesis. We show here that Translesion synthesis (TLS) and DNA-damage-induced mu- η ζ human Pol inserts a nucleotide opposite the lesion, followed tagenesis are the best-characterized functions of Pol . Absence by Pol ζ extending the DNA primer; thus, the two complement REV3 of the yeast gene leads to sensitivity to UV light and each other to fully bypass the cisplatin cross-link. intrastrand and interstrand cross-linking agents (2, 15). DNA Pol ζ has been shown to induce multiple base substitutions as well as Author contributions: Y.-S.L. and W.Y. designed research; Y.-S.L. and M.T.G. performed re- more complex mutations in yeast (7, 16, 17) and may contribute search; Y.-S.L., M.T.G., and W.Y. analyzed data; and Y.-S.L. and W.Y. wrote the paper. to hypermutation in Ig genes in mammals (18, 19). The TLS The authors declare no conflict of interest. ζ function of DNA Pol has been implicated in its role of medi- 1Present address: Well-Aging Research Center, Samsung Advanced Institute of Technol- ating resistance to platinum-based chemotherapies (20–22). Owing ogy, Samsung Electronics, Yongin, Gyeonggido, Korea. E-mail: [email protected]. to the conservation of B-family DNA polymerases, a distorted 2To whom correspondence should be addressed. E-mail: [email protected]. DNA template base is unlikely to be accommodated in the active This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. site of DNA Pol ζ. In fact, yeast DNA Pol ζ is unable to insert 1073/pnas.1324001111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1324001111 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 Purification and characterization of Pol ζ has so far been Scrutinizing the primary structure of the inserted sequences, we limited to the yeast protein. Perhaps because of its large size, identified a positively charged domain (PCD; 960–1,200 aa), mammalian Pol ζ has not been purified for biochemical char- which is ubiquitously present between the NTD and R7B among acterization. To overcome this roadblock, we coexpressed hu- vertebrate Rev3 orthologs. Accordingly, we constructed expres- man REV3L and REV7 in mammalian cells in culture. Initially, sion vectors of the full-length human REV3L and six variants very low expression level and heterogeneity was encountered, but with deletions of the predicted unstructured sequences that these problems were solved by targeted deletion of various in- surround the three conserved domains and the PCD (Fig. 1A). ternal segments of human REV3L. We succeeded in purifying an His8 and maltose binding protein (MBP) tags were placed in active two-subunit form of human Pol ζ (Pol ζ2). By differential tandem on the N terminus of Rev3 or Rev7 to increase protein pull-down experiments using Pol ζ2 variants with and without the solubility and simplify protein purification. The seven REV3L CTD of Rev3, we isolated two CTD-dependent Pol ζ accessary constructs were individually cotransfected with REV7 into HEK293 subunits, PolD2 and PolD3. We report here purification of an cells for transient protein expression. Pol ζ2 was partially purified active form of human four-subunit Pol ζ4 and the collaboration from harvested cells by using an amylose affinity column (Fig. of two TLS polymerases, Pol η and Pol ζ, in lesion bypass. 1B and Fig. S1). When full-length Rev3 was MBP tagged, only trace amounts of a degraded MBP-fusion protein of ∼110 kDa Results was detected by Western blot (Fig. S1A). Even with a Rev3 Coexpression and Purification of Human Rev3 and Rev7. Two major deletion variant, the yield of Pol ζ2 was 10 times greater when insertions in human Rev3 separate three highly conserved domains: Rev7 was tagged instead of Rev3 (Fig. S1B). Therefore, we chose the N-terminal domain (NTD; 1–333 aa), the Rev7-binding domain to tag Rev7 and not Rev3. (R7B; 1,888–1,943 aa), and the C-terminal polymerase domain Coexpression and purification of the full-length Rev3 and (Pol; 2,276–3,130 aa) (ref. 4; Fig. 1A). Most of the inserted MBP–Rev7 yielded low amounts and low purity of human Pol sequences (∼1,500 aa) are predicted to be random coil inter- ζ (Fig. S2A). In addition to the 353-kDa band of the full-length spersed with low complexity segments (bioinf.cs.ucl.ac.uk/psipred). hRev3, an equal intensity band of ∼300 kDa was observed, which

Fig. 1. Determination of a minimal Rev3 that forms active human Pol ζ.(A) Alignment of the conserved domains between yeast and human Rev3. The NTD is shaded in blue, R7B in yellow, and the Pol in red. Domain boundaries are indicated by residue numbers. The positively charged domain (PCD) shaded in green is unique to vertebrate Rev3. In human Rev3 deletion constructs TR1, TR3, TR4-2, TR4-3, TR4-5, and TR5, predicted unstructured portions with boundaries marked by residue numbers were deleted and replaced by two amino acids as indicated. (B) SDS gel stained by Coo- massie Blue R-250 of purified Rev3–Rev7 (Pol ζ2) variants. Each variant partially purified by amylose column was loaded at an equal amount (2.5 μg) and labeled according to the Rev3 construct. MBP–Rev7 was more abundant than Rev3 in each case. (C) DNA synthesis by each of six Pol ζ2 variants. Equal proportions of Pol ζ2 proteins as loaded in the SDS gel (in B) were used in the DNA synthesis assay. The DNA primers were 5′-FAM Fluorescein-labeled. (D) Activity comparison of Pol ζ2 variants. The amount of Rev3 in each lane in B (representing active Pol ζ2) and the amount of the full-length DNA products (43 and 44 nt) in C were quantified based on the band intensity. Data for the full-length Rev3 are shown in Fig. S1.The specific activity of each Pol ζ2 variant (the amount of DNA product divided by the amount of Rev3 protein) was calculated and then normalized to that of TR4-2.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1324001111 Lee et al. Downloaded by guest on September 28, 2021 likely corresponds to a large fragment of Rev3 (see below). With a single internal deletion of residues 527–1,800 in Rev3 (TR1), the yield of Pol ζ increased by ∼10-fold (Fig. 1 A and B). However, the specific polymerase activity of TR1 on a normal DNA template–primer pair was ∼2% that of full- length Pol ζ (Fig. 1 and Fig. S2). Inclusion of the positively charged patch (1,042–1,254 aa) and alteration of boundaries surrounding the R7B domain (TR3, TR4-2, and TR4-3) resulted in an approximately eightfold specific-activity increase of TR4-2 relative to TR1 without reducing the protein yield (Fig. 1). Comparison among TR1, TR3, TR4-2, and TR4-3 constructs suggested that inclusion of amino acids 1,588–1,800 and deletion of amino acids 2,048–2,217 enhanced the protein yield and specific activity. The most significant increase in the Pol ζ-specific activity was achieved by the addition of residues 900–1,041 (TR4-5 and TR5) (Fig. 1), which restored the specific catalytic activity of TR5 (a total of 2,358 aa) to that of Fig. 2. Identification of human Pol ζ accessary subunits. (A) A diagram of ζ the full-length Rev3 (3,130 aa). However, this activity increase Pol and potential accessary subunits. (B) Pull-down of Rev3 accessary sub- was accompanied by a significant decrease in protein yield and units from HEK293 cells. The TR4-2 variants of Rev3 with or without CTD were constructed and coexpressed with MBP–Rev7 as baits to pull down Pol the appearance of a proteolyzed Rev3 band (Fig. 1B). The size ζ — 2-interacting proteins. Bands 1 and 2 were purified only in the presence of difference between the expected Rev3 whether full-length, the CTD. Band 2 overlapped with band x, which was copurified with Rev3 TR4-5, or TR5—and the proteolyzed bands on SDS gels with or without the CTD. (C) Mass spectrometry analysis identified bands 1 appeared to be constant. We therefore estimate that the cleav- and 2 as PolD3 (p66) and PolD2 (p50), respectively, and band x as tubulins. age site is around residue 500 and suspect that the presence of residues 900–1,041 somehow alters the Rev3 structure to expose this protease cleavage site. For the remaining Pol ζ studies tag), and PolD3 (p66 with or without a GST tag) (Fig. 3 A–C) reported here, we focus on the TR4-2 variant (2,113 of 3,130 aa into HEK293 cells. To ensure that no endogenous PolD2 and

total), which produces a single Rev3 band on SDS gels and an PolD3 copurified with Pol ζ2, a TR4-2ΔCTD clone was used to BIOCHEMISTRY ζ generate Pol ζ2. For the negative control in enzymatic assays, we active Pol 2 that has the same nucleotide preference and gen- − erates the same DNA products as the full-length Rev3 (Fig. S2). generated and purified inactive Pol ζ4 protein by mutating two catalytically essential aspartates in Rev3 to asparagines (D2614N Identification of Human DNA Pol ζ Accessory Subunits. The con- and D2783N). Differently tagged Pol ζ4 proteins (MBP-tagged served Cys-rich CTDs of B-family DNA polymerases α, δ, and e p50 or GST-tagged p66) were purified by amylose or amylose each interact with unique accessory subunits. To determine and glutathione affinity columns, followed by cation-exchange − whether the CTD of human Rev3 (3,016–3,130 aa) also interacts chromatography. Pol ζ2, ζ4, and ζ4 were equally well expressed with its own accessory proteins, we constructed a CTD-deletion and equally stable (Fig. 3). Interestingly, PolD2 (p50) or PolD3 clone of TR4-2 (TR4-2ΔCTD) and cotransfected expression (p66) that was untagged—and thus might suffer loss during the — vectors of TR4-2 with or without CTD and His8–MBP–Rev7 into affinity purification was present in less than stoichiometric HEK293 cells (Fig. 2A). To ensure that the amount of Rev3 and amounts in the purified four-subunit complex Pol ζ4 (Fig. 3 B Rev7 did not greatly exceed endogenous Pol ζ cofactors, we and C). The varying ratios of PolD2 and PolD3 in the human Pol reduced the DNA amounts used for transfection. Without as- ζ4 preparations when different subunits were tagged suggest that suming which proteins might interact with the CTD of Pol ζ,we PolD3 may interact with human Rev3 or Rev7 independent of fractionated the total HEK293 cell lysate with an amylose pull- the PolD2–CTD interaction (23, 37, 38). down followed by a cation exchange column. The eluents were To compare the Pol ζ catalytic activities, equimolar concen- − treated with PreScission protease to separate Rev7 from the trations of Pol ζ2, ζ4, and ζ4 proteins were tested in normal His –MBP tag and analyzed on a SDS gel with Coomassie blue DNA synthesis assays (Fig. 3 B–D). Although negligible, residual 8 − stain (Fig. 2B). The presence of the Rev3 CTD resulted in two DNA synthesis activity was detected with the Pol ζ4 protein additional pull-down protein bands. Band 1 (∼65 kDa) was (Fig. 3 B–E), which could be due to a trace contaminant with completely absent in the lane without the Rev3 CTD, and active DNA polymerases—for example, Pol δ or Rev1 copur- band 2 (∼50 kDa) ran very closely to a protein band (band x) ifying via interaction with PolD2 or Rev7. Even with PolD2 or present in the lane without the Rev3 CTD (Fig. 2B). With >55% PolD3 in substoichiometric amounts relative to Rev3 (TR4-2), of each protein band sequenced by mass spectrometry, bands 1 Pol ζ4 exhibited higher kcat and lower KM than Pol ζ2 (Fig. 3D) and 2 were identified as human PolD3 (p66) and PolD2 (p50), and was nearly 30 times more efficient than Pol ζ2 in catalyzing homologs of yeast Pol32 and Pol31, respectively. Band x was DNA synthesis in the absence of a lesion. identified to be α and β tubulins. The other protein band (band y) The presence of PolD2 and PolD3 accessory subunits also pulleddownwithorwithouttheRev3CTDwas∼75 kDa and increased the processivity of human Pol ζ4 on DNA substrates. is too small to be Rev1, which is known to associate weakly Full-length DNA primer-extension products (42- to 44-nt bands) with Rev7 (39–42). Based on our prior experience with protein were prominent with Pol ζ4 but very weak with Pol ζ2 (Fig. 3 B expression in HEK293 cells, band y is likely a protein chap- and C). To semiquantitatively assess the processivity of Pol ζ4 erone. We conclude that, like yeast Pol ζ,humanPolζ is a and Pol ζ2, the primer extension assay was performed with four-subunit DNA polymerase and shares two accessory subunits a range of concentrations of the two protein complexes, and with DNA Pol δ. primer use and product distributions were then compared (Fig. 3E). At the lowest concentration of Pol ζ4 and highest concen- PolD2 and PolD3 Enhance the Catalytic Efficiency and Processivity of tration of Pol ζ2, for which nearly 50% of DNA primers were Pol ζ. To investigate how PolD2 and PolD3 influence the stability extended in each case, Pol ζ4 produced at least five times more and catalytic activity of human Pol ζ, we prepared Pol ζ2 and Pol full-length DNA products than Pol ζ2. The increased processivity ζ4 by cotransfection of two or four relevant expression vectors of was likely due to enhanced DNA binding by human Pol ζ4, which TR4-2, His8–MBP–Rev7, PolD2 (p50 with or without an MBP may also explain the reduced KM.

Lee et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 − Fig. 3. DNA synthesis by human Pol ζ2 and ζ4. (A) Diagrams of Pol ζ4(Left), ζ2(Center), and ζ4 (Right), which were inactivated by mutations in the polymerase active site (D2614N/D2783N). (B) Protein preparation (Left) and DNA synthesis (Right) assay of the Pol ζ4 with both Rev7 and PolD2 MBP-tagged. The amount of MBP–PolD2 (p50) exceeded the untagged PolD3 (p66), which ran slightly faster than MBP–Rev7. With equal amount of Rev3, Pol ζ4 was much more efficient than Pol ζ2 in producing the full-length DNA products. (C) Protein preparation (Left) and DNA synthesis (Right) assay of the Pol ζ4 with GST- tagged p66. The amount of GST–PolD3 (p66) exceeded the untagged PolD2 (p50). The DNA synthesis activities of the two differently tagged Pol ζ4 were comparable, indicating that all four subunits were essential, but the reduced amount of an accessary subunit might limit the apparent activity of Pol ζ4. (D)

Measurement of KM and kcat of Pol ζ2 and ζ4 in dATP incorporation opposite a normal dT template base (P25–T44 DNA). (E) Comparison of the Pol ζ2 and ζ4 processivity. Dilution series of Pol ζ2 (35, 70, and 140 nM) and Pol ζ4 (13, 35 and 70 nM) were used in the primer extension assay. Both the primer use and the full-length extension (42∼44 nt) products were quantified and are indicated below each lane.

Pol ζ and Pol η Synergistically Bypass an Intrastrand Cisplatin Cross- A, T, or G opposite a template G in the normal or Pt-GG DNA Link. Human Pol ζ has been implicated in resistance to platinum- (Fig. 4B), although the degree of nucleotide discrimination will based chemotherapies (20–22), and knockdown of REV3L sensi- require further quantitative studies. tizes malignant cells to cisplatin treatment (21, 43). We compared In our previous studies of Pt-GG bypass, we found that human the catalytic activity and accuracy of Pol ζ4 in synthesizing DNA in Y-family Pol η (1-432aa) is both accurate and efficient in inserting the presence or absence of a 1,2-intrastrand d(GpG)-cisplatin a C opposite the first G of a Pt-GG lesion, but its catalytic ef- cross-link (abbreviated as Pt-GG hereafter). The relative catalytic ficiency decreases steeply in subsequent primer extension (44). efficiencies of Pol ζ4 in nucleotide incorporation opposite a cross- The opposite trends of Pol ζ4 and Pol η in catalytic efficiency of linked Pt-GG and in primer extension after the lesion were sep- bypassing Pt-GG suggest that they may complement each other arately assayed at each step by using four primers of different and synergize to carry out TLS. Indeed, when the two poly- lengths paired with two template strands of the same sequence— merases were combined, Pt-GG bypass starting opposite the le- one with Pt-GG and the other without (Fig. 4A). Pol ζ4 was in- sion was far more efficient and more complete than when using active when encountering the 3′ GofPt-GG(thefirstoftwocross- either polymerase alone (Fig. 4C). Notably, Pol η had low linked GG) and failed to insert any nucleotide in the presence of processivity on both damaged and normal DNA, and Pol ζ4 had individual or all four nucleotides. Pol ζ4, however, accurately lower catalytic activity but higher processivity than Pol η. Pol η inserted a C opposite the 5′ G of Pt-GG (the second of the cross- was able to assist Pol ζ4 to overcome the Pt-GG block (with the linked GG). The efficiency of primer extension after Pt-GG by Pol P23 primer) as well as stalling in the middle of primer extension ζ4 increased step by step with increased kcat and decreased KM opposite undamaged DNA (Fig. 4C). (Table 1). With a normal base pair between the template and primer after the Pt-GG lesion, primer extension by Pol ζ4reached Discussion 10–15% of the catalytic efficiency and >95% of kcat observed in We succeeded in expressing and purifying an active form of the absence of Pt-GG (Table 1). A contaminant 3′-exonuclease human Pol ζ, which enabled us to carry out the previously im- activity was observed when the P26 primer was paired with the possible in vitro characterization of Pol ζ. Mammalian Rev3 is Pt-GG template (Fig. 4B), and the resulting P26 degradation was twice as large as the yeast homolog. Among the 1,500 residues lesion dependent and Pol ζ4 activity independent (Fig. S3). (500∼1,800 and 2,000∼2,200 aa) of Rev3 that are unique in Consistent with its error-prone reputation, Pol ζ4 misincorporated higher eukaryotes, we identified a 340-residue highly positively

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1324001111 Lee et al. Downloaded by guest on September 28, 2021 Fig. 4. Pol ζ4andPolη carry out TLS synergistically bypassing a Pt-GG lesion. (A) Diagrams of DNA primer and template used in the assay. The four primers allow separate analyses of nucleotide insertions opposite the Pt-GG dimer and primer extension 1 or 2 nt beyond the lesion. The template strands contain either cisplatin cross-linked Pt-GG (in red) or undamaged DNA of the same sequence. (B) Primer extension of undamaged and Pt-GG DNA template by Pol ζ4 in the presence of each (A, T, G, or C) or all four nucleotides (labeled as 4). Percentage of each primer extended in the presence of a correct dNTP or mixed four nucleotides was quantified and is indicated. (Upper) Insertion step. (Lower) Extension step. (C)Polζ4andPolη were synergistic in DNA synthesis. (Upper) With normal DNA template, Pol η was efficient in nucleotide incorporation but had poor processivity. (Lower) In the presence of a Pt-GG lesion, Pol ζ4 failed to insert opposite the 3′ G of the Pt-GG, whereas Pol η failed to extend after the Pt-GG lesion. Together, Pol ζ4andPolη resulted in 10% of full-length product. BIOCHEMISTRY

charged domain (residues 960–1,200), which contains low-com- only with Pol31 (38). However, the recent EM study of yeast plexity sequence dominated by Lys (17.8%), Ser (10%), and Arg Pol ζ4 placed Pol32 proximal to Rev7 (46). Potential inter- (8.3%). Its inclusion enhances the specific activity of DNA syn- actions between human PolD3 (yeast Pol32 homolog) and thesis by Pol ζ. The exact amino acid sequence of PCD is not Rev7 may explain our observation that PolD3 remains a com- highly conserved, but the Lys- and Arg-rich character is con- ponent of Pol ζ4, even when PolD3 is in molar excess of PolD2 served from invertebrates to mammals, suggesting that charge– (Fig. 3C). Unless PolD2 and PolD3 are limiting in vivo, both charge interactions may lead to increased DNA binding by Pol ζ. Pol δ and ζ may exist as a four-subunit complex without com- Our deletion analysis also revealed that the region between R7B peting for the accessory units. (Rev7 binding) and the polymerase catalytic domain (residues In human Pol ζ4, the presence of PolD2 and PolD3, even at 2,048–2,217) negatively impacts the polymerase activity of Pol ζ2 submolar ratios to Rev3 (TR4-2), dramatically increased the (Fig. 1). The remaining 770 residues, which can be removed from efficiency (kcat/KM) and processivity of DNA synthesis (Fig. 3D). human Rev3 without reducing the polymerase activity of Pol ζ2 The effect of decreased KM and increased processivity suggests as shown by our TR-5 construct, may play a role in interactions that PolD2 and PolD3 significantly enhance association of Pol with other factors to regulate the activity of Pol ζ and its re- ζ with DNA substrates. The cocrystal structure of human cruitment to a replication fork. PolD2 and the NTD of PolD3 has been determined (35) and Based on studies of yeast Pol ζ and the CTD of human Rev3, it shows a primarily negatively charged molecular surface with- is not surprising that human PolD2 and PolD3, the equivalents of out obvious features for DNA binding. It is possible that the yeast Pol31 and Pol32, are accessory subunits of human Pol ζ. unstructured, but positively charged, region in PolD3 that is Our pull-down experiments using whole-cell lysates of HEK293 not included in the crystal structure may play a crucial role in cells, however, were conducted without the assumption of enhancing the Pol ζ catalytic activity and processivity. In PolD2 and PolD3 involvement. Only PolD2 and PolD3 co- contrast, inclusion of two accessory subunits in yeast Pol ζ4 purified with Rev3 (TR4-2) and Rev7 after stringent washing primarily increases the kcat by twofold to threefold without an with a 1.0 M NaCl solution, and Rev1, a known cofactor of Pol appreciable change of KM or processivity (23). This difference ζ via interactions with Rev7 (41, 42, 45), was not detected between yeast and human Pol ζ4 may result from low similarity (Fig. 2B). PolD2 and PolD3 must have stronger interactions between human PolD3 and yeast Pol32 accessory subunits. with Rev3 and Rev7 than Rev1. In yeast, Pol31 directly Pol ζ and Pol η are synergistic in improving the efficiency and interacts with the CTD of Pol ζ and Pol δ, and Pol32 interacts processivity of DNA synthesis both in the absence and presence

Table 1. Catalytic efficiency of Pol ζ4 in normal DNA synthesis and Pt-GG bypass Incoming Relative efficiency, Relative rate, −1 −1 −1 Primer Template nucleotide kcat,min KM, μM kcat/KM,M ·min %ofkcat/Km %ofkcat

P24 (second insertion) Undamaged dCTP 0.22 ± 0.01 1.50 ± 0.12 144.7 x 103 100 100 Pt-GG 0.09 ± 0.01 55.8 ± 10.4 1.7 x 103 1.2 43.3 P25 (first extension) Undamaged dATP 0.31 ± 0.01 1.17 ± 0.08 261.5 x 103 100 100 Pt-GG 0.15 ± 0.01 35.6 ± 4.2 4.1 x 103 1.6 47.7 P26 (second extension) Undamaged dGTP 0.22 ± 0.01 0.82 ± 0.09 268.3 x 103 100 100 Pt-GG 0.21 ± 0.01 8.80 ± 0.97 24.1 x 103 9.0 96.4

Lee et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 of a DNA lesion (Fig. 4C). In the absence of direct interactions Materials and Methods ζ η between Pol and Pol , the synergy most likely takes place by Human Pol ζ2 and ζ4 with His8–MBP-labeled Rev7 were expressed in tran- passing DNA substrate between the two polymerases. Pol η and siently transfected HEK293 cells and purified by using affinity pull-down, Pol ζ appear to complement each other in catalytic efficiency ion-exchange, and size-exclusion chromatography. The catalytically inactive − and processivity in both TLS, as demonstrated here and in main- Pol ζ4 mutant was generated by using QuikChange (Stratagene). Catalytic tenance of common fragile sites in human genome as reported activities of Pol ζ variants were measured with Fluorescein (6-FAM)-labeled (14, 47). However, without necessary regulation of their access to primers as described (26). A full description of materials and methods can be DNA replication forks, the substantial efficiency and processivity found in SI Materials and Methods. of these two low-fidelity DNA polymerases could lead to a high frequency of mutagenesis. We expect that stringent down-regulation ACKNOWLEDGMENTS. We thank Dr. Christopher W. Lawrence for human ζ REV3L and MAD2L2 cDNAs and Drs. R. Craigie and D. J. Leahy for editing this of Pol may keep this essential polymerase engaged only when manuscript. This work is supported by National Institutes of Health Intra- necessary for lesion bypass and cell proliferation. mural Research Grant DK036146-07.

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