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Stability of the Human Polymerase Δ Holoenzyme and Its Implications in Lagging Strand DNA Synthesis

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Stability of the Human Polymerase Δ Holoenzyme and Its Implications in Lagging Strand DNA Synthesis Stability of the human polymerase δ holoenzyme and PNAS PLUS its implications in lagging strand DNA synthesis Mark Hedglina, Binod Pandeya, and Stephen J. Benkovica,1 aDepartment of Chemistry, The Pennsylvania State University, University Park, PA 16802 Edited by Jerard Hurwitz, Memorial Sloan-Kettering Cancer Center, New York, NY, and approved February 17, 2016 (received for review November 30, 2015) In eukaryotes, DNA polymerase δ (pol δ) is responsible for replicat- the front face of the clamp is oriented toward the 3′ end of the ing the lagging strand template and anchors to the proliferating cell nascent primer/template (P/T) junction where DNA synthesis nuclear antigen (PCNA) sliding clamp to form a holoenzyme. The will initiate. An incoming pol δ subsequently captures the stability of this complex is integral to every aspect of lagging strand loaded PCNA ring, forming a holoenzyme, and DNA synthesis replication. Most of our understanding comes from Saccharomyces initiates (2, 5). cerevisae where the extreme stability of the pol δ holoenzyme en- The stability of the lagging strand holoenzyme is integral to sures that every nucleobase within an Okazaki fragment is faithfully various aspects of Okazaki fragment synthesis. For eukaryotes, duplicated before dissociation but also necessitates an active dis- most of our understanding comes from studies in Saccharomyces placement mechanism for polymerase recycling and exchange. cerevisae, where the three-subunit pol δ is extremely stable with However, the stability of the human pol δ holoenzyme is unknown. −3 −1 PCNA on DNA (koff < 2 × 10 s , t1/2 > 5 min). Once DNA We designed unique kinetic assays to analyze the processivity and synthesis is initiated from a nascent primer, the dramatically slow stability of the pol δ holoenzyme. Surprisingly, the results indicate k ensures every nucleotide within a given Okazaki fragment is that human pol δ maintains a loose association with PCNA while off faithfully duplicated before dissociation. On the other hand, such replicating DNA. Such behavior has profound implications on Okazaki fragment synthesis in humans as it limits the processivity of pol δ on high stability necessitates an active mechanism for displacement of δ pol δ once DNA synthesis stops (6, 7). This situation arises when a undamaged DNA and promotes the rapid dissociation of pol from δ ′ PCNA on stalling at a DNA lesion. pol holoenzyme encounters either the 5 RNA end of a down- stream Okazaki fragment (pol recycling) or distortions to the BIOCHEMISTRY lagging strand | stability | PCNA | DNA polymerase delta | native sequence that it cannot accommodate (pol exchange), such translesion DNA synthesis as common byproducts of UV radiation exposure (8). Pol recy- cling allows the scarce pol δ to be reused during S-phase, whereas uring S-phase of the cell cycle, genomic DNA must be pol exchange permits a specialized pol to bind to PCNA and faithfully copied in a short period. Replicative DNA poly- synthesize past the offending damage [translesion DNA syn- D δ – merases (pols) alone are distributive and must anchor to ring- thesis (TLS)] so that pol may resume synthesis (9 12). How- shaped sliding clamps to achieve the high degree of processivity ever, studies on the human pol δ holoenzyme are lacking, and required for efficient DNA replication. The highly conserved to- hence, the mechanisms by which polymerase recycling and ex- roidal structure of sliding clamps has a central cavity large enough change occur are unknown. To gain insight, we designed a to encircle double-stranded DNA (dsDNA) and slide freely along unique kinetic assay to measure the stability of the pol δ ho- it. Thus, such an association effectively tethers the pol to DNA, loenzyme. Surprisingly, the results indicate that human pol δ substantially increasing the extent of continuous replication. The maintains a loose association with PCNA. Such behavior has eukaryotic sliding clamp, proliferating cell nuclear antigen (PCNA), profound implications on lagging strand synthesis as it limits is trimer of identical subunits aligned head-to-tail, forming a ring the extent of processive DNA synthesis and promotes the rapid with two structurally distinct faces. Each subunit consists of two dissociation of pol δ from PCNA on stalling. independent domains connected by an interdomain connecting “ ” loop (IDCL). The front face of the homotrimeric PCNA ring Significance contains all IDCLs and is a platform for interaction with the eukaryotic replicative pols, e and δ, which are responsible for the The results from the reported studies indicate that the human faithful replication of the leading and lagging strands, respec- lagging strand polymerase, pol δ, maintains a loose association tively (1, 2). Specifically, the well-conserved PCNA-interacting with the sliding clamp, proliferating cell nuclear antigen (PCNA), peptide (PIP) box within replicative pols makes extensive contact while replicating and rapidly dissociates on stalling, leaving PCNA with an IDCL of PCNA and displays conserved residues that behind on the DNA. This behavior has profound implications on “plug” into the proximal hydrophobic patches. The amino acid lagging strand synthesis as it limits the extent of processive DNA sequence of a canonical PIP box is QXXhXXaa, where X rep- synthesis on undamaged DNA and promotes the rapid dissociation resents any amino acid, h is a hydrophobic residue (usually L, I, of pol δ on stalling at a replication-blocking lesion. This challenges or M), and a is an aromatic residue (usually F or Y) (3). the accepted models for polymerase recycling and exchange on the Unlike the leading strand, the lagging strand is synthesized lagging strand and instead suggests passive mechanisms for the discontinuously in short Okazaki fragments that are processed human system. These studies provide valuable insight for future and ligated together to form a continuous strand (4). In eu- experiments in the fields DNA replication, DNA repair, and DNA karyotes, each Okazaki fragment is initiated by the bifunctional damage tolerance. DNA pol α/primase complex that lays down cRNA/DNA hybrid – primers every 100 250 nucleotides (nt) on the exposed tem- Author contributions: M.H. and S.J.B. designed research; M.H. and B.P. performed re- plate for the lagging strand. The intermittent single-stranded search; M.H., B.P., and S.J.B. analyzed data; and M.H. and S.J.B. wrote the paper. DNA (ssDNA) is protected from cellular nucleases by repli- The authors declare no conflict of interest. cation protein A (RPA), a ssDNA binding protein that also This article is a PNAS Direct Submission. prevents formation of alternative DNA structures. The clamp 1To whom correspondence should be addressed. Email: [email protected]. loader, replication factor C (RFC), recognizes these hybrid This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. primers abutted by RPA and loads PCNA onto each such that 1073/pnas.1523653113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1523653113 PNAS | Published online March 14, 2016 | E1777–E1786 Downloaded by guest on September 26, 2021 amino acid (13–21). The homotrimeric PCNA sliding clamp contains three identical binding sites for PIP box-containing pro- teins and, hence, human pol δ may simultaneously bind all three subunits within a given PCNA trimer, similar to that observed in S. cerevisae (22). Indeed, sequential removal of the p12 and p66 Fig. 1. Sequence of the biotinylated primer/template P29/Bio62. The size of accessory subunits from the human pol δ assembly reduced the the double-stranded P/T region (29 bp) is in agreement with the size of an initiating hybrid P/T and the requirements for assembly of a human pol δ extent of PCNA-dependent DNA synthesis in a stepwise manner, holoenzyme by RFC (5, 70). The biotin attached to the 3′-end of the template suggesting that all PCNA-interacting subunits are required to form strand was prebound to neutravidin, preventing loaded PCNA from sliding off a holoenzyme with optimal DNA synthetic activity (23). In all the 5′ end of the primer. The single-stranded DNA (ssDNA) adjacent to the 3′ experiments discussed herein, only the complete, four-subunit end of the P/T junction was prebound with excess RPA. The size of this region human pol δ complex was used. Furthermore, pol exchange in (33 nt) is consistent with the size of ssDNA covered by a single RPA molecule eukaryotes involves the conjugation of single ubiquitin moieties – δ (22 30 nt) (71). To monitor extension of the primer by pol within a single (i.e., monoubiquitination) to lysine residues (K164) of PCNA re- binding encounter, the 29-mer primer was 32P-end-labeled before annealing and reactions were carried out in the presence of a passive DNA trap. In all siding at a stalled P/T junction. This posttranslational modification experiments, the trap is unlabeled substrate lacking a biotin tag and con- (PTM) is essential for optimal TLS activity in mammalian cells taining a 3′-dideoxy-terminated primer (referred to as 29ddC/62). (24), but its role in pol exchange is under intense debate. To gain insight into a potential role of this PTM involving the lagging strand pol δ, we synthesized monoubiquitinated PCNA [referred Results to herein as (Ub)3-PCNA] that contains a single ubiquitin moiety Monitoring a Single Turnover of Primer Extension by Human pol δ. on K164 of each monomer within a homotrimeric clamp ring (25). Human pol δ is comprised of four subunits: three accessory sub- The conjugation of ubiquitin to PCNA has no effect on the in- units (p50/POLD2, p66/POLD3, and p12/POLD4) and a large teraction of PCNA with RFC (Fig. S1B) or on the ability of RFC catalytic subunit (p125/POLD1) that contains both DNA poly- to load PCNA onto DNA (Fig. S2), in agreement with observa- merase and exonuclease domains.
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