scholarly journals Structure of eukaryotic DNA polymerase δ bound to the PCNA clamp while encircling DNA

2020 ◽  
Vol 117 (48) ◽  
pp. 30344-30353
Author(s):  
Fengwei Zheng ◽  
Roxana E. Georgescu ◽  
Huilin Li ◽  
Michael E. O’Donnell
Keyword(s):  
Dna Polymerase ◽  
Human Cancer ◽  
Catalytic Subunit ◽  
Nuclear Antigen ◽  
Genome Replication ◽  
Regulatory Subunits ◽  
Minimum Drag ◽  
Disease Mutations ◽  
Eukaryotic Dna ◽  
Health And Disease

The DNA polymerase (Pol) δ ofSaccharomyces cerevisiae(S.c.) is composed of the catalytic subunit Pol3 along with two regulatory subunits, Pol31 and Pol32. Pol δ binds to proliferating cell nuclear antigen (PCNA) and functions in genome replication, repair, and recombination. Unique among DNA polymerases, the Pol3 catalytic subunit contains a 4Fe-4S cluster that may sense the cellular redox state. Here we report the 3.2-Å cryo-EM structure of S.c. Pol δ in complex with primed DNA, an incoming ddTTP, and the PCNA clamp. Unexpectedly, Pol δ binds only one subunit of the PCNA trimer. This singular yet extensive interaction holds DNA such that the 2-nm-wide DNA threads through the center of the 3-nm interior channel of the clamp without directly contacting the protein. Thus, a water-mediated clamp and DNA interface enables the PCNA clamp to “waterskate” along the duplex with minimum drag. Pol31 and Pol32 are positioned off to the side of the catalytic Pol3-PCNA-DNA axis. We show here that Pol31-Pol32 binds single-stranded DNA that we propose underlies polymerase recycling during lagging strand synthesis, in analogy toEscherichia colireplicase. Interestingly, the 4Fe-4S cluster in the C-terminal CysB domain of Pol3 forms the central interface to Pol31-Pol32, and this strategic location may explain the regulation of the oxidation state on Pol δ activity, possibly useful during cellular oxidative stress. Importantly, human cancer and other disease mutations map to nearly every domain of Pol3, suggesting that all aspects of Pol δ replication are important to human health and disease.

scholarly journals Structure of the processive human Pol δ holoenzyme

2019 ◽  
Author(s):  
Claudia Lancey ◽  
Muhammad Tehseen ◽  
Vlad-Stefan Raducanu ◽  
Fahad Rashid ◽  
Nekane Merino ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Structural Basis ◽  
Catalytic Core ◽  
Regulatory Subunits ◽  
Functional Interactions ◽  
Proliferating Cell ◽  
Lagging Strand ◽  
Catalytic Cleft

In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. We present the high-resolution cryo-EM structure of the human processive Pol δ-DNA-PCNA complex in the absence and presence of FEN1. Pol δ is anchored to one of the three PCNA monomers through the C-terminal domain of the catalytic subunit. The catalytic core sits on top of PCNA in an open configuration while the regulatory subunits project laterally. This arrangement allows PCNA to thread and stabilize the DNA exiting the catalytic cleft and recruit FEN1 to one unoccupied monomer in a toolbelt fashion. Alternative holoenzyme conformations reveal important functional interactions that maintain PCNA orientation during synthesis. This work sheds light on the structural basis of Pol δ’s activity in replicating the human genome.

scholarly journals Mutant DNA polymerase δ from thermosensitive Schizosaccharomyces pombe strains display reduced stimulation by proliferating cell nuclear antigen

1998 ◽  
Vol 335 (3) ◽  
pp. 581-588 ◽  
Author(s):  
Mylène PERDERISET ◽  
Giovanni MAGA ◽  
Karine PIARD ◽  
Stefania FRANCESCONI ◽  
Isabelle TRATNER ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Catalytic Subunit ◽  
Nuclear Antigen ◽  
Wild Type ◽  
Low Levels ◽  
Proliferating Cell ◽  
Stimulation Of

We have isolated and characterized DNA polymerase δ (pol δ) from two thermosensitive Schizosaccharomyces pombe strains, polδts1 and polδts3, mutated in two different evolutionarily conserved domains of the catalytic subunit. At the restrictive temperature of 37 °C polδts1 and polδts3 mutant strains arrest growth in the S phase of the cell cycle. We show that at low levels of primer ends, in vitro stimulation by proliferating cell nuclear antigen (PCNA) of mutant enzymes is lower than stimulation of wild-type pol δ. Affinity for primer (3´-OH) ends and processivity of mutant enzymes do not appear different from wild-type pol δ. In contrast, Vmax values are lower than the wild-type value. The major in vitro defect appears to be decreased stimulation of mutant enzymes by PCNA, resulting in reduced velocity of DNA synthesis. In addition, ts1 pol δ is not stimulated by low PCNA concentration at 37 °C, although low concentrations stimulate activity at 25 °C, suggesting that this thermolability at low levels of primer ends could be its critical defect in vivo. Thus, both ts1 and ts3 pol δ mutations are located in regions of the catalytic subunit that seem necessary, directly or indirectly, for its efficient interaction with PCNA.

scholarly journals The tamas Gene, Identified as a Mutation That Disrupts Larval Behavior in Drosophila melanogaster, Codes for the Mitochondrial DNA Polymerase Catalytic Subunit (DNApol-γ125)

Genetics ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 1809-1824 ◽  
Author(s):  
Balaji Iyengar ◽  
John Roote ◽  
Ana Regina Campos
Keyword(s):  
Mitochondrial Dna ◽  
Drosophila Melanogaster ◽  
Mutant Strain ◽  
Dna Polymerase ◽  
Complementation Group ◽  
Behavioral Changes ◽  
Catalytic Subunit ◽  
Primary Deficit ◽  
Mitochondrial Dna Polymerase ◽  
Food Substrate

AbstractFrom a screen of pupal lethal lines of Drosophila melanogaster we identified a mutant strain that displayed a reproducible reduction in the larval response to light. Moreover, this mutant strain showed defects in the development of the adult visual system and failure to undergo behavioral changes characteristic of the wandering stage. The foraging third instar larvae remained in the food substrate for a prolonged period and died at or just before pupariation. Using a new assay for individual larval photobehavior we determined that the lack of response to light in these mutants was due to a primary deficit in locomotion. The mutation responsible for these phenotypes was mapped to the lethal complementation group l(2)34Dc, which we renamed tamas (translated from Sanskrit as “dark inertia”). Sequencing of mutant alleles demonstrated that tamas codes for the mitochondrial DNA polymerase catalytic subunit (DNApol-γ125).

scholarly journals A Mutation of the Yeast Gene Encoding PCNA Destabilizes Both Microsatellite and Minisatellite DNA Sequences

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 511-519 ◽  
Author(s):  
Robert J Kokoska ◽  
Lela Stefanovic ◽  
Andrew B Buermeyer ◽  
R Michael Liskay ◽  
Thomas D Petes
Keyword(s):  
Dna Polymerase ◽  
Repetitive Dna ◽  
Dna Sequences ◽  
Repeat Unit ◽  
Nuclear Antigen ◽  
Temperature Sensitive ◽  
Dinucleotide Repeats ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Polymerase Δ ◽  
Repeat Units

AbstractThe POL30 gene of the yeast Saccharomyces cerevisiae encodes the proliferating cell nuclear antigen (PCNA), a protein required for processive DNA synthesis by DNA polymerase δ and ϵ. We examined the effects of the pol30-52 mutation on the stability of microsatellite (1- to 8-bp repeat units) and minisatellite (20-bp repeat units) DNA sequences. It had previously been shown that this mutation destabilizes dinucleotide repeats 150-fold and that this effect is primarily due to defects in DNA mismatch repair. From our analysis of the effects of pol30-52 on classes of repetitive DNA with longer repeat unit lengths, we conclude that this mutation may also elevate the rate of DNA polymerase slippage. The effect of pol30-52 on tracts of repetitive DNA with large repeat unit lengths was similar, but not identical, to that observed previously for pol3-t, a temperature-sensitive mutation affecting DNA polymerase δ. Strains with both pol30-52 and pol3-t mutations grew extremely slowly and had minisatellite mutation rates considerably greater than those observed in either single mutant strain.

scholarly journals Archaeal DNA Polymerase D but Not DNA Polymerase B Is Required for Genome Replication in Thermococcus kodakarensis

2013 ◽  
Vol 195 (10) ◽  
pp. 2322-2328 ◽  
Author(s):  
L. Cubonova ◽  
T. Richardson ◽  
B. W. Burkhart ◽  
Z. Kelman ◽  
B. A. Connolly ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Genome Replication ◽  
Thermococcus Kodakarensis

A gene-sized DNA molecule encoding the catalytic subunit of DNA polymerase a in the macronucleus of Oxytricha nova

Gene ◽  
1994 ◽  
Vol 144 (2) ◽  
pp. 155-161 ◽  
Author(s):  
Sam J. Mansour ◽  
David C. Huffman ◽  
David M. Prescott
Keyword(s):  
Dna Polymerase ◽  
Catalytic Subunit ◽  
Dna Molecule ◽  
Oxytricha Nova
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