The C-terminal Domain of the DNA Polymerase Catalytic Subunit Regulates the Primase and Polymerase Activities of the Human DNA Polymerase α-Primase Complex

Zhang, Yinbo; Baranovskiy, Andrey G.; Tahirov, T.H.; Pavlov, Youri I.

doi:10.1074/jbc.m114.570333

Cited by 33 publications

(55 citation statements)

References 60 publications

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“…Sequence of the biotinylated primer/template P29/Bio62. The size of 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 holoenzyme by RFC (5,70). The biotin attached to the 3′-end of the template strand was prebound to neutravidin, preventing loaded PCNA from sliding off the 5′ end of the primer.…”

Section: Resultsmentioning

confidence: 58%

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: 58%

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 NTD of the B subunit of Pol ⑀, which has a similar structure (51), is important for the assembly of Cdc45-MCM-GINS helicase and integration of Pol ⑀ into the eukaryotic replisome during initiation (26,52). In Prim-Pol ␣, the CTD-B subunit complex plays an additional and unique role by tethering two catalytic domains and regulating their activities (53).…”

Section: Discussionmentioning

confidence: 99%

Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit

Suwa¹,

Gu²,

Baranovskiy³

et al. 2015

Journal of Biological Chemistry

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Background: DNA polymerase ␣ (Pol ␣) initiates DNA synthesis and is indispensable for genome replication.Results: We present a crystal structure of human Pol ␣ minus the catalytic core at 2.5 Å resolution. Conclusion:The mode of interaction between the Pol ␣ subunits is evolutionarily conserved. Significance: The data provide structural insight into the function of the primase-Pol ␣ complex.

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“…In humans, Prim consists of the small catalytic subunit (p49; also known as p48, PRIM1, Pri1, and PriS) and the large regulatory subunit (p58; also known as PRIM2, Pri2, and PriL), whereas Pol ␣ is composed of the catalytic subunit (p180) and the accessory subunit (p70). The evolutionarily conserved C-terminal domain of p180 (p180 C ) plays an important structural role by tethering both p70 and primase to the Pol ␣ catalytic subunit (2)(3)(4)(5). The tight physical association of primase with DNA polymerase, found only in eukaryotes, ensures effective intramolecular substrate transfer between the two active sites.…”

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confidence: 99%

“…It has also been proposed that the large subunit helps to "count" the product size and participates in translocation of the primertemplate to the Pol ␣ active site (10,12). The N-terminal part of the large subunit (p58 N in human primase) provides a platform for interactions with p49 and Pol ␣ (5,16). Most structural elements contributing to NTP and DNA binding by the large subunit are located in the C-terminal half of the protein (p58 C in human primase) (11,(17)(18)(19)(20).…”

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confidence: 99%

“…The tight physical association of primase with DNA polymerase, found only in eukaryotes, ensures effective intramolecular substrate transfer between the two active sites. When primase synthesizes the unit-length RNA primers (7-10 nucleotides long), the primed template is translocated to the Pol ␣ active site and is extended by 20 -30 deoxynucleotides (5)(6)(7)(8).…”

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confidence: 99%

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Crystal Structure of the Human Primase

Baranovskiy

Zhang

Suwa

et al. 2015

Journal of Biological Chemistry

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101

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The C-terminal Domain of the DNA Polymerase Catalytic Subunit Regulates the Primase and Polymerase Activities of the Human DNA Polymerase α-Primase Complex

Cited by 33 publications

References 60 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

Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit

Crystal Structure of the Human Primase

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