PCNA binding domains in all three subunits of yeast DNA polymerase δ modulate its function in DNA replication

Acharya, Narottam; Klassen, Roland; Johnson, Robert E.; Prakash, Louise; Prakash, Satya

doi:10.1073/pnas.1109981108

Cited by 75 publications

(94 citation statements)

References 29 publications

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“…The p66 accessory subunit contains a canonical PIP box, whereas the p125 catalytic subunit and the p12 accessory subunit both contain noncanonical PIP domains where the conserved Q residue has been replaced with an alternative amino acid (13)(14)(15)(16)(17)(18)(19)(20)(21). The homotrimeric PCNA sliding clamp contains three identical binding sites for PIP box-containing proteins 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 accessory subunits from the human pol δ assembly reduced the extent of PCNA-dependent DNA synthesis in a stepwise manner, suggesting that all PCNA-interacting subunits are required to form a holoenzyme with optimal DNA synthetic activity (23).…”

Section: Resultsmentioning

confidence: 85%

“…Hence, the probability of insertion for i = 2 is equal to the fraction of pol δ holoenzymes remaining. (B) 16% denaturing sequencing gel of the primer extension products for pol δ holoenzymes assembled with either PCNA (lanes 1-13) or (Ub) 3 -PCNA (lanes [14][15][16][17][18][19][20][21][22][23][24][25][26]. The size of the substrate and full-length product is indicated on the left.…”

Section: Resultsmentioning

confidence: 99%

“…For example, the probability that a primer extension product 46 nt in length will be extended to a product of 55 nt in length, a total of nine insertion steps, is equal to (P 18-26 ) 9 . To calculate (P [18][19][20][21][22][23][24][25][26], the concentration of all primer extension products at least 55 nt in length was divided by the concentration of all primer extension products at least 46 nt in length, and the ninth root of this ratio reflects the average probability of insertion (P [18][19][20][21][22][23][24][25][26] ) for steps i = 18 through i = 26. Pol δ Holoenzyme Stability Assays.…”

Section: Methodsmentioning

confidence: 99%

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

Hedglin

Pandey

Benkovic

2016

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultsmentioning

confidence: 85%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Hedglin

Pandey

Benkovic

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…The p50 subunit also binds to PCNA, but very weakly [Wang et al, 2011], and, in further discussions, we will consider Pol d as having three operational-binding motifs for PCNA. The three subunits of yPold3 all interact with PCNA, and each of their binding motifs contributes to processivity when assayed in vitro [Acharya et al, 2011], even though they are individually dispensable for short-term survival. The presence of multiple-binding interactions may provide conformational flexibility, whereby Pol d may bind to PCNA via multiple conformations without necessarily engaging the primer terminus.…”

Section: Roles Of Pol D3 In Translesion Synthesis By Tls Polymerasesçmentioning

confidence: 99%

Regulation of human DNA polymerase delta in the cellular responses to DNA damage

Lee

Zhang

Lin

et al. 2012

Environ and Mol Mutagen

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The p12 subunit of polymerase delta (Pol d) is degraded in response to DNA damage induced by UV, alkylating agents, oxidative, and replication stresses. This leads to the conversion of the Pol d4 holoenzyme to the heterotrimer, Pol d3. We review studies that establish that Pol d3 formation is an event that could have a major impact on cellular processes in genomic surveillance, DNA replication, and DNA repair. p12 degradation is dependent on the apical ataxia telangiectasia and Rad3 related (ATR) kinase and is mediated by the ubiquitin-proteasome system. Pol d3 exhibits properties of an ''antimutator'' polymerase, suggesting that it could contribute to an increased surveillance against mutagenesis, for example, when Pol d carries out bypass synthesis past small base lesions that engage in spurious base pairing. Chromatin immunoprecipitation analysis and examination of the spatiotemporal recruitment of Pol d to sites of DNA damage show that Pol d3 is the primary form of Pol d associated with cyclobutane pyrimidine dimer lesions and therefore should be considered as the operative form of Pol d engaged in DNA repair. We propose a model for the switching of Pol d with translesion polymerases, incorporating the salient features of the recently determined structure of monoubiquitinated proliferating cell nuclear antigen and emphasizing the role of Pol d3. Because of the critical role of Pol d activity in DNA replication and repair, the formation of Pol d3 in response to DNA damage opens the prospect that pleiotropic effects may ensue. This opens the horizons for future exploration of how this novel response to DNA damage contributes to genomic stability. Environ. Mol. Mutagen. 53:683-698, 2012. V V C 2012 Wiley Periodicals, Inc.

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“…Two additional motifs, one in Pol3 and one in Pol31, were recently reported, emphasizing that ScPol d has multiple surfaces to stabilize the interaction with PCNAwhen synthesizing DNA (Acharya et al 2011). Less is known about how Pol 1 interacts with PCNA.…”

Section: Eukaryamentioning

confidence: 99%

Replicative DNA Polymerases

Johansson

Dixon

2013

Cold Spring Harbor Perspectives in Biology

109

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In 1959, Arthur Kornberg was awarded the Nobel Prize for his work on the principles by which DNA is duplicated by DNA polymerases. Since then, it has been confirmed in all branches of life that replicative DNA polymerases require a single-stranded template to build a complementary strand, but they cannot start a new DNA strand de novo. Thus, they also depend on a primase, which generally assembles a short RNA primer to provide a 3 0 -OH that can be extended by the replicative DNA polymerase. The general principles that (1) a helicase unwinds the double-stranded DNA, (2) single-stranded DNA-binding proteins stabilize the single-stranded DNA, (3) a primase builds a short RNA primer, and (4) a clamp loader loads a clamp to (5) facilitate the loading and processivity of the replicative polymerase, are well conserved among all species. Replication of the genome is remarkably robust and is performed with high fidelity even in extreme environments. Work over the last decade or so has confirmed (6) that a common two-metal ion-promoted mechanism exists for the nucleotidyltransferase reaction that builds DNA strands, and (7) that the replicative DNA polymerases always act as a key component of larger multiprotein assemblies, termed replisomes. Furthermore (8), the integrity of replisomes is maintained by multiple protein-protein and protein -DNA interactions, many of which are inherently weak. This enables large conformational changes to occur without dissociation of replisome components, and also means that in general replisomes cannot be isolated intact.

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PCNA binding domains in all three subunits of yeast DNA polymerase δ modulate its function in DNA replication

Cited by 75 publications

References 29 publications

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

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

Regulation of human DNA polymerase delta in the cellular responses to DNA damage

Replicative DNA Polymerases

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