The eukaryotic leading and lagging strand DNA polymerases are loaded onto primer-ends via separate mechanisms but have comparable processivity in the presence of PCNA

In eukaryotes, DNA polymerase δ (pol δ) is responsible for replicating the lagging strand template and anchors to the proliferating cell nuclear antigen (PCNA) sliding clamp to form a holoenzyme. The stability of this complex is integral to every aspect of lagging strand replication. Most of our understanding comes from Saccharomyces cerevisae where the extreme stability of the pol δ holoenzyme ensures that every nucleobase within an Okazaki fragment is faithfully duplicated before dissociation but also necessitates an active displacement mechanism for polymerase recycling and exchange. However, the stability of the human pol δ holoenzyme is unknown. We designed unique kinetic assays to analyze the processivity and stability of the pol δ holoenzyme. Surprisingly, the results indicate that human pol δ maintains a loose association with PCNA while replicating DNA. Such behavior has profound implications on Okazaki fragment synthesis in humans as it limits the processivity of pol δ on undamaged DNA and promotes the rapid dissociation of pol δ from PCNA on stalling at a DNA lesion.lagging strand | stability | PCNA | DNA polymerase delta | translesion DNA synthesis D uring S-phase of the cell cycle, genomic DNA must be faithfully copied in a short period. Replicative DNA polymerases (pols) alone are distributive and must anchor to ringshaped sliding clamps to achieve the high degree of processivity required for efficient DNA replication. The highly conserved toroidal structure of sliding clamps has a central cavity large enough to encircle double-stranded DNA (dsDNA) and slide freely along it. Thus, such an association effectively tethers the pol to DNA, substantially increasing the extent of continuous replication. The eukaryotic sliding clamp, proliferating cell nuclear antigen (PCNA), is trimer of identical subunits aligned head-to-tail, forming a ring with two structurally distinct faces. Each subunit consists of two independent domains connected by an interdomain connecting loop (IDCL). The "front" face of the homotrimeric PCNA ring contains all IDCLs and is a platform for interaction with the eukaryotic replicative pols, e and δ, which are responsible for the faithful replication of the leading and lagging strands, respectively (1, 2). Specifically, the well-conserved PCNA-interacting peptide (PIP) box within replicative pols makes extensive contact with an IDCL of PCNA and displays conserved residues that "plug" into the proximal hydrophobic patches. The amino acid sequence of a canonical PIP box is QXXhXXaa, where X represents any amino acid, h is a hydrophobic residue (usually L, I, or M), and a is an aromatic residue (usually F or Y) (3).Unlike the leading strand, the lagging strand is synthesized discontinuously in short Okazaki fragments that are processed and ligated together to form a continuous strand (4). In eukaryotes, each Okazaki fragment is initiated by the bifunctional DNA pol α/primase complex that lays down cRNA/DNA hybrid primers every 100-250 nucleotides (nt) on the exposed template for the l...

Section: Resultssupporting

confidence: 83%

Stability of the human polymerase δ holoenzyme and its implications in lagging strand DNA synthesis

Hedglin

Pandey

Benkovic

2016

“…In fact, human Pol δ is known to be distributive with PCNA (25), and barring stability by another protein, quality control on the leading strand may be explained by spontaneous dissociation in humans. We note that different processivity values of S. cerevisiae Pol δ-PCNA are reported, which probably depend on the substantially different ionic strengths used in the studies, but could also possibly be due to different methods of protein preparation (12,13). It is important to note that the intracellular salt in yeast is not yet identified, nor has the intracellular ionic strength of S. cerevisiae been determined.…”

Section: Quality Control Of Polmentioning

confidence: 89%

“…However, our earlier studies made the perplexing observation that Pol e was incapable of lagging strand synthesis even in the absence of Pol δ (9, 10). We and others have shown that S. cerevisiae Pol δ-PCNA is rapid, over 100 bp/s, and its processivity varies depending on ionic strength (12,13). The intracellular ionic strength of S. cerevisiae is unknown, but at ionic strength under 70 mM, Pol δ-PCNA is highly processive for over 5 kb (13).…”

mentioning

confidence: 87%

See 1 more Smart Citation

Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork

Schauer

O'Donnell

2017

The eukaryotic genome is primarily replicated by two DNA polymerases, Pol e and Pol δ, that function on the leading and lagging strands, respectively. Previous studies have established recruitment mechanisms whereby Cdc45-Mcm2-7-GINS (CMG) helicase binds Pol e and tethers it to the leading strand, and PCNA (proliferating cell nuclear antigen) binds tightly to Pol δ and recruits it to the lagging strand. The current report identifies quality control mechanisms that exclude the improper polymerase from a particular strand. We find that the replication factor C (RFC) clamp loader specifically inhibits Pol e on the lagging strand, and CMG protects Pol e against RFC inhibition on the leading strand. Previous studies show that Pol δ is slow and distributive with CMG on the leading strand. However, Saccharomyces cerevisiae Pol δ-PCNA is a rapid and processive enzyme, suggesting that CMG may bind and alter Pol δ activity or position it on the lagging strand. Measurements of polymerase binding to CMG demonstrate Pol e binds CMG with a K d value of 12 nM, but Pol δ binding CMG is undetectable. Pol δ, like bacterial replicases, undergoes collision release upon completing replication, and we propose Pol δ-PCNA collides with the slower CMG, and in the absence of a stabilizing Pol δ-CMG interaction, the collision release process is triggered, ejecting Pol δ on the leading strand. Hence, by eviction of incorrect polymerases at the fork, the clamp machinery directs quality control on the lagging strand and CMG enforces quality control on the leading strand.D uplication of genetic material is performed by a dynamic interplay of numerous different proteins, collectively referred to as the replisome, that orchestrate their actions to accomplish efficient, high-fidelity replication of both the leading and lagging strands (1-3). Eukaryotes possess several replisome factors that have no homologs in bacteria, and also require two different DNA polymerases for bulk leading and lagging strand synthesis, and Escherichia coli and its phages use identical copies of a DNA polymerase for both strands of the duplex (4). Thus far, the evolutionary purpose behind the additional complexity in eukaryotes is unclear.At the heart of the eukaryotic replisome is an 11-subunit helicase complex referred to as CMG (Cdc45-Mcm2-7-GINS) (5-7). Eukaryotes use three different multisubunit B-family DNA polymerases (Pol) for replication. Pol e and Pol δ copy the bulk of the genome, and Pol α-primase generates hybrid RNA-DNA primers of about 25 nucleotides and, unlike Pol e and Pol δ, it lacks 3′-5′ exonuclease (proofreading) activity. The replication factor C (RFC) clamp loader is used to load PCNA (proliferating cell nuclear antigen) clamps onto primed sites that endow the replicative polymerases with high processivity (8). The replication protein A (RPA) heterotrimer binds and protects the single-strand (ss) DNA of the lagging strand against nucleases, and helps melt secondary structure in ssDNA. Reconstitution of the core eukaryotic replisome using all thre...

“…A second explanation is that PCNA trimers with more WT binding sites may have an intrinsically superior ability to stimulate partner enzymes. To examine this possibility, we performed true processivity assays (26), which measure the rate of PCNA-Pol δ dissociation after each binding event (Fig. S8).…”

Section: Discussionmentioning

confidence: 99%

Sequential switching of binding partners on PCNA during in vitro Okazaki fragment maturation

Dovrat

Stodola

Burgers

et al. 2014

The homotrimeric sliding clamp proliferating cell nuclear antigen (PCNA) mediates Okazaki fragment maturation through tight coordination of the activities of DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (Lig1). Little is known regarding the mechanism of partner switching on PCNA and the involvement of PCNA's three binding sites in coordinating such processes. To shed new light on PCNA-mediated Okazaki fragment maturation, we developed a novel approach for the generation of PCNA heterotrimers containing one or two mutant monomers that are unable to bind and stimulate partners. These heterotrimers maintain the native oligomeric structure of PCNA and exhibit high stability under various conditions. Unexpectedly, we found that PCNA heterotrimers containing only one functional binding site enable Okazaki fragment maturation by efficiently coordinating the activities of Pol δ, FEN1, and Lig1. The efficiency of switching between partners on PCNA was not significantly impaired by limiting the number of available binding sites on the PCNA ring. Our results provide the first direct evidence, to our knowledge, that simultaneous binding of multiple partners to PCNA is unnecessary, and if it occurs, does not provide significant functional advantages for PCNA-mediated Okazaki fragment maturation in vitro. In contrast to the "toolbelt" model, which was demonstrated for bacterial and archaeal sliding clamps, our results suggest a mechanism of sequential switching of partners on the eukaryotic PCNA trimer during DNA replication and repair.