Structure of eukaryotic DNA polymerase δ bound to the PCNA clamp while encircling DNA Fengwei Zhenga, Roxana E. Georgescub,c, Huilin Lia,1, and Michael E. O’Donnellb,c,1 aStructural Biology Program, Van Andel Institute, Grand Rapids, MI 49503; bDNA Replication Laboratory, The Rockefeller University, New York, NY 10065; and cHHMI, The Rockefeller University, New York, NY 10065 Contributed by Michael E. O’Donnell, October 9, 2020 (sent for review August 20, 2020; reviewed by David Jeruzalmi and Zvi Kelman) The DNA polymerase (Pol) δ of Saccharomyces cerevisiae (S.c.) is optimally with the replicative CMG helicase to which it binds composed of the catalytic subunit Pol3 along with two regulatory (11, 12). Pol δ performs lagging strand synthesis by extending subunits, Pol31 and Pol32. Pol δ binds to proliferating cell nuclear primers generated by Pol α primase every 100 to 200 nt (9). Pol δ antigen (PCNA) and functions in genome replication, repair, and also synthesizes a small amount of leading strand DNA during recombination. Unique among DNA polymerases, the Pol3 cata- replication initiation and termination (13, 14). Both Pol δ and lytic subunit contains a 4Fe-4S cluster that may sense the cellular Pol e function with the PCNA sliding clamp (9, 15). In fact, Pol δ redox state. Here we report the 3.2-Å cryo-EM structure of S.c. Pol has little activity in the absence of PCNA, which stimulates Pol δ δ in complex with primed DNA, an incoming ddTTP, and the PCNA activity by a factor of 30 (16). clamp. Unexpectedly, Pol δ binds only one subunit of the PCNA The sliding clamps of bacteria and eukaryotes are structurally trimer. This singular yet extensive interaction holds DNA such that superimposable and are also utilized in numerous processes be- the 2-nm-wide DNA threads through the center of the 3-nm inte- yond replication, including use by translesion synthesis (TLS) Pols, rior channel of the clamp without directly contacting the protein. and a multitude of different repair factors, such as DNA ligase, Thus, a water-mediated clamp and DNA interface enables the Fen1 nuclease, MutS, and MutL to mention only a few, making PCNA clamp to “waterskate” along the duplex with minimum sliding clamps an attractive therapeutic target (15, 17). Sliding drag. Pol31 and Pol32 are positioned off to the side of the catalytic clamps are homo-oligomers, and each subunit contains a hydro- Pol3-PCNA-DNA axis. We show here that Pol31-Pol32 binds single- phobic pocket to which proteins attach (1, 17). Thus, clamps have stranded DNA that we propose underlies polymerase recycling the potential to simultaneously bind multiple partners, acting like during lagging strand synthesis, in analogy to Escherichia coli rep- a “tool belt.” In eukaryotes, the motif that proteins use to attach to BIOCHEMISTRY licase. Interestingly, the 4Fe-4S cluster in the C-terminal CysB do- the clamp’s hydrophobic pocket is referred to as a PCNA inter- main of Pol3 forms the central interface to Pol31-Pol32, and this action peptide (PIP) motif, and a similar motif is utilized by strategic location may explain the regulation of the oxidation proteins that attach to the bacterial β clamp (18). The canonical state on Pol δ activity, possibly useful during cellular oxidative PIP motif is QxxΨxxθθ (where Ψ is hydrophobic, θ is aromatic, and stress. Importantly, human cancer and other disease mutations x is any residue). There are also noncanonical PIP sequences that map to nearly every domain of Pol3, suggesting that all aspects differ widely from the canonical PIP motif (19). of Pol δ replication are important to human health and disease. This report focuses on the Saccharomyces cerevisiae (S.c.) replicative Pol δ and its interaction with the PCNA clamp. S.c. BIOPHYSICS AND COMPUTATIONAL BIOLOGY DNA polymerase | sliding clamp | PCNA | DNA polymerase δ | DNA Pol δ contains three subunits. Pol3 harbors the catalytic Pol and replication proofreading 3′-5′ exonuclease, and the regulatory subunits are Pol31 (i.e., the B subunit) and Pol32. Human Pol δ contains an eplication of the cellular DNA genome is accomplished by RDNA polymerases (Pol) that function with a ring shaped Significance “sliding clamp” (1–3). The sliding clamp encircles duplex DNA and tethers the Pol to DNA for high processivity during synthesis The structure of the eukaryotic chromosomal replicase, DNA (4, 5). Sliding clamps are assembled onto primed-template (T/P) polymerase (Pol) δ, was determined in complex with its cog- junctions by a pentameric clamp loader that couples ATP nate proliferating cell nuclear antigen (PCNA) sliding clamp on binding and hydrolysis to opening and closing of the ring around primed DNA. The results show that the Pol3 catalytic subunit DNA (6). All three domains of life utilize an evolutionarily con- binds atop the PCNA ring, and the two regulatory subunits of served sliding clamp and clamp loader; in eukaryotes/archaea, Pol δ, Pol31, and Pol32, are positioned off to the side of the Pol3 these are the proliferating cell nuclear antigen (PCNA) clamp and clamp. The catalytic Pol3 binds DNA and PCNA such as to thread replication factor C (RFC), and in bacteria they are referred to as the DNA straight through the circular PCNA clamp. Considering the β clamp and γ complex (7). DNA polymerases assort into the large diameter of the PCNA clamp, there is room for water seven families by sequence homology—families A, B, C, D, X, Y, between DNA and the inner walls of PCNA, indicating the and reverse transcriptase—although structural analysis shows that clamp “waterskates” on DNA during function with polymerase. all DNA polymerases are shaped like a right hand and contain subdomains referred to as palm, fingers, and thumb (8). The Author contributions: H.L. and M.E.O. designed research; F.Z. and R.E.G. performed re- cellular replicative polymerases of the three domains of life belong search; F.Z., R.E.G., H.L., and M.E.O. analyzed data; and F.Z., R.E.G., H.L., and M.E.O. wrote the paper. to the C family (bacteria), the B family (eukaryotes), or the B and Reviewers: D.J., City College of New York; and Z.K., National Institute of Standards and D family (archaea). Technology. Eukaryotes utilize three essential B family DNA polymerases The authors declare no competing interest. for genome replication: Pol α, Pol δ, and Pol e (9). Each of these This open access article is distributed under Creative Commons Attribution License 4.0 B family DNA polymerases is highly conserved from yeast to (CC BY). α α human (9). Pol , referred to as Pol -primase, generates 20- to 1To whom correspondence may be addressed. Email: [email protected] or odonnel@ 30-nt-long RNA-DNA primers to initiate the synthesis of leading rockefeller.edu. and lagging strands. Pols δ and e are the main replicative poly- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ merases, and each contain a proofreading 3′-5′ exonuclease. Pol doi:10.1073/pnas.2017637117/-/DCSupplemental. e replicates the bulk of the leading strand (9, 10) and functions www.pnas.org/cgi/doi/10.1073/pnas.2017637117 PNAS Latest Articles | 1of10 Downloaded by guest on September 24, 2021 additional regulatory subunit, Pol12 (9). The exact functions of inactive phosphodiesterase and polymerase domain (PDE), some- the “regulatory subunits” are not well defined. Unique to DNA times referred to as a polymerase and histidinol phosphatase polymerases, the catalytic Pol3 subunit of Pol δ contains a 4Fe- (PHP) domain, which might interact with downstream single-strand 4S iron sulfur cluster (20). During lagging strand synthesis, Pol δ (ss) DNA (Fig. 1 C and D). Indeed, we found that isolated Pol31- is capable of releasing its PCNA clamp soon after completing Pol32 bound to ssDNA in electrophoretic mobility shift assays synthesis of a DNA gap (i.e., Okazaki fragment) (21), similar to (EMSAs) (SI Appendix,Fig.S3). The PDE/PHP domain is also observations in the Escherichia coli replicase system (22, 23). In present in bacterial Pol III and is an active proofreading 3′-5′ addition, Pol δ-PCNA functions with the Fen1 5′ nuclease on a exonuclease in some Pol IIIs while being inactive in other Pol IIIs millisecond time scale to strand-displace and excise the 5′ RNA- that have recruited a separate 3′-5′ exonuclease subunit/domain DNA of Okazaki fragments, followed by DNA ligase I (Lig1) (35). Active PDE/PHE domains consist of several α helices in a that seals the nick (16). This rapid timescale suggests that Fen1 barrel conformation and harbor catalytic metals coordinated by and Lig1 bind PCNA simultaneously with Pol δ for this frequent nine conserved residues; the inactive E. coli Pol III PDE/PHP Okazaki fragment maturation reaction. Indeed, an archaeal domain has lost five metal-binding residues during evolution (SI PCNA is a heterotrimer in which each clamp subunit binds a Appendix,Fig.S4A and B). The PDE/PHP domain of Pol δ has lost separate factor, Pol D, Fen1, or ligase. Indeed, PCNA in the six metal binding residues, and the barrel shape is less compact and archaeon Sulfolobus solfataricus is a heterotrimer in which each somewhat distorted. However, a human disease mutation maps to clamp subunit binds a separate factor: Pol B, Fen1, or ligase this location, suggesting the PDE/PHP domain is important to Pol (24, 25). δ function (SI Appendix,TableS2). Pol δ has a far greater role beyond its role in DNA replication. While this work was ongoing, the structure of human Pol For example, Pol δ, along with Pol η, is required for lesion bypass δ-PCNA-DNA was reported (32); it has the same overall fea- δ on both leading- and lagging-strand DNA (26).