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