The DNA Polymerase _-Primase Complex: Multiple Functions and Interactions

Muzi-Falconi, Marco; Giannattasio, Michele; Foiani, Marco; Plevani, Paolo

doi:10.1100/tsw.2003.05

Cited by 59 publications

(53 citation statements)

References 61 publications

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“…Pol α is the only polymerase in eukaryotic cells that can synthesize primers de novo on ssDNA templates, owing to the enzyme's dual primase and DNA polymerase activities. 18 Pol α performs two important functions during unperturbed replication; one, it initiates replication on the leading strand and two, it continuously initiates Okazaki fragment synthesis during lagging strand replication. Previous studies have also linked pol α's primase activity to ATR activation.…”

Section: Introductionmentioning

confidence: 99%

TopBP1 and DNA polymerase alpha-mediated recruitment of the 9-1-1 complex to stalled replication forks: Implications for a replication restart-based mechanism for ATR checkpoint activation

Shan¹,

Michael²

2009

Cell Cycle

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Here, we summarize recent advances in understanding the multiple roles played by TopBP1 in ATR activation at stalled replication forks. We review recent studies showing that TopBP1 controls the loading of 9-1-1 onto stalled replication forks via a pathway that also requires DNA polymerase alpha (pol α). Based on these recent studies, we present a revised model for ATR activation, and speculate that TopBP1-mediated recruitment of pol α and 9-1-1 may couple checkpoint activation to replication restart, when DNA synthesis is blocked on the leading strand of a replication fork. Lastly, we present a new experiment that examines how TopBP1 binds to stalled replication forks, and we identify important new questions that our recent studies have raised regarding how stalled replication forks are sensed by the ATR checkpoint pathway.

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Section: Introductionmentioning

confidence: 99%

TopBP1 and DNA polymerase alpha-mediated recruitment of the 9-1-1 complex to stalled replication forks: Implications for a replication restart-based mechanism for ATR checkpoint activation

Shan¹,

Michael²

2009

Cell Cycle

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“…Thus, we speculate that a hydrophobic interaction at the OB-fold may provide additional specificity for p180 286 -310 . Pol ␣ and Mcm10 Binding Domains-The overall domain structure of p180 is known, and the activities of the central polymerase and the C-terminal subunit assembly domains have been characterized (53,54). However, the function of the N-terminal domain is less clear.…”

Section: Chemical Nature Of Mcm10-id Interactions With Dna Andmentioning

confidence: 99%

Physical Interactions between Mcm10, DNA, and DNA Polymerase α

Warren

Huang²,

Fanning³

et al. 2009

Journal of Biological Chemistry

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Mcm10 is an essential eukaryotic protein required for the initiation and elongation phases of chromosomal replication. Specifically, Mcm10 is required for the association of several replication proteins, including DNA polymerase ␣ (pol ␣), with chromatin. We showed previously that the internal (ID) and C-terminal (CTD) domains of Mcm10 physically interact with both single-stranded (ss) DNA and the catalytic p180 subunit of pol ␣. However, the mechanism by which Mcm10 interacts with pol ␣ on and off DNA is unclear. As a first step toward understanding the structural details for these critical intermolecular interactions, x-ray crystallography and NMR spectroscopy were used to map the binary interfaces between Mcm10-ID, ssDNA, and p180. The crystal structure of an Mcm10-ID⅐ssDNA complex confirmed and extended our previous evidence that ssDNA binds within the oligonucleotide/oligosaccharide binding-fold cleft of Mcm10-ID. We show using NMR chemical shift perturbation and fluorescence spectroscopy that p180 also binds to the OB-fold and that ssDNA and p180 compete for binding to this motif. In addition, we map a minimal Mcm10 binding site on p180 to a small region within the p180 N-terminal domain (residues 286 -310). These findings, together with data for DNA and p180 binding to an Mcm10 construct that contains both the ID and CTD, provide the first mechanistic insight into how Mcm10 might use a handoff mechanism to load and stabilize pol ␣ within the replication fork.

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“…In eukaryotes, the primosome, a tight complex of DNA primase and DNA polymerase ␣ (Pol␣), 4 synthesizes primers for both leading and lagging strands in a highly coordinated fashion (1,2). The primosome is indispensable for initiation of replication and has a large impact on genome stability (3)(4)(5)(6).…”

mentioning

confidence: 99%

Mechanism of Concerted RNA-DNA Primer Synthesis by the Human Primosome

Baranovskiy

Babayeva

Zhang

et al. 2016

Journal of Biological Chemistry

116

228

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The human primosome, a 340-kilodalton complex of primase and DNA polymerase ␣ (Pol␣), synthesizes chimeric RNA-DNA primers to be extended by replicative DNA polymerases ␦ and ⑀. The intricate mechanism of concerted primer synthesis by two catalytic centers was an enigma for over three decades. Here we report the crystal structures of two key complexes, the human primosome and the C-terminal domain of the primase large subunit (p58 C ) with bound DNA/RNA duplex. These structures, along with analysis of primase/polymerase activities, provide a plausible mechanism for all transactions of the primosome including initiation, elongation, accurate counting of RNA primer length, primer transfer to Pol␣, and concerted autoregulation of alternate activation/inhibition of the catalytic centers. Our findings reveal a central role of p58 C in the coordinated actions of two catalytic domains in the primosome and ultimately could impact the design of anticancer drugs.In eukaryotes, the primosome, a tight complex of DNA primase and DNA polymerase ␣ (Pol␣), 4 synthesizes primers for both leading and lagging strands in a highly coordinated fashion (1, 2). The primosome is indispensable for initiation of replication and has a large impact on genome stability (3-6). RNA primer synthesis by primase involves three steps: initiation, elongation, and termination (7,8). During the rate-limiting initiation step, primase binds the DNA template and two ribonucleotide triphosphates (NTPs) and catalyzes the formation of a dinucleotide (9, 10). Further synthesis of the RNA primer is much faster but restricted, because of the intrinsic property of primases to count the primer length and terminate synthesis after incorporation of 8 -10 nucleotides (7). Next, the mature so-called "unit length" RNA primer is intramolecularly translocated to Pol␣ for the subsequent extension by dNTPs, and the primase became inhibited by an unknown mechanism (9,11,12). Orchestration of all these steps requires changes in primosome conformation (13).Human Pol␣ (Fig. 1A) is comprised of a large catalytic subunit (p180) and a smaller accessory subunit (p70), connected by the C-terminal domain of p180 (p180 C ) containing two conserved zinc-binding modules, Zn1 and Zn2 (14 -16). p70 consists of an N-terminal (p70 N ), a phosphodiesterase, and oligonucleotide/oligosaccharide-binding (OB) domains (14, 17). The globular p70 N is attached to the phosphodiesterase via a flexible linker (amino acid residues 79 -156) (14, 18) and participates in interactions with other DNA replication proteins (19). The catalytic core of p180 (p180core) and p180 C -p70 are connected by a 15-residue linker (1251-1265) (13). Human primase consists of catalytic (p49) and regulatory (p58) subunits (20). p58 has two distinct domains, N-terminal (p58 N ) and C-terminal (p58 C ), connected with an 18-residue linker (253-270) (21). p58 N interacts with p49 and connects primase with Pol␣ (22, 23), and an iron-sulfur cluster containing p58 C plays an important role in substrate binding and primase acti...

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The DNA Polymerase _-Primase Complex: Multiple Functions and Interactions

Cited by 59 publications

References 61 publications

TopBP1 and DNA polymerase alpha-mediated recruitment of the 9-1-1 complex to stalled replication forks: Implications for a replication restart-based mechanism for ATR checkpoint activation

TopBP1 and DNA polymerase alpha-mediated recruitment of the 9-1-1 complex to stalled replication forks: Implications for a replication restart-based mechanism for ATR checkpoint activation

Physical Interactions between Mcm10, DNA, and DNA Polymerase α

Mechanism of Concerted RNA-DNA Primer Synthesis by the Human Primosome

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