Trading Places on DNA—A Three-Point Switch Underlies Primer Handoff from Primase to the Replicative DNA Polymerase

Yuzhakov, A. A.; Kelman, Zvi; O’Donnell, Michael

doi:10.1016/s0092-8674(00)80968-x

Cited by 211 publications

(211 citation statements)

References 31 publications

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“…In the case of E. coli, regulation of this process resides in the C-terminal region of the polymerase accessory subunit . Upon reaching the nick resulting from a completed Okazaki fragment, it displaces the ␤-clamp from the polymerase subunit ␣ (30), and a set of three step switches delivers the polymerase to the next primer (31). During primer delivery, the binding of the polymerase subunit to the helicase occurs via a highly basic, flexible domain at the C terminus of (28).…”

Section: Discussionmentioning

confidence: 99%

A unique loop in T7 DNA polymerase mediates the binding of helicase-primase, DNA binding protein, and processivity factor

Hamdan

Marintcheva

Cook

et al. 2005

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

150

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Bacteriophage T7 DNA polymerase (gene 5 protein, gp5) interacts with its processivity factor, Escherichia coli thioredoxin, via a unique loop at the tip of the thumb subdomain. We find that this thioredoxin-binding domain is also the site of interaction of the phage-encoded helicase͞primase (gp4) and ssDNA binding protein (gp2.5). Thioredoxin itself interacts only weakly with gp4 and gp2.5 but drastically enhances their binding to gp5. The acidic C termini of gp4 and gp2.5 are critical for this interaction in the absence of DNA. However, the C-terminal tail of gp4 is not required for binding to gp5 when the latter is bound to a primer͞template. We propose that the thioredoxin-binding domain is a molecular switch that regulates the interaction of T7 DNA polymerase with other proteins of the replisome.DNA replication ͉ molecular switch ͉ replisome ͉ gene 4 protein ͉ thioredoxin

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Section: Discussionmentioning

confidence: 99%

A unique loop in T7 DNA polymerase mediates the binding of helicase-primase, DNA binding protein, and processivity factor

Hamdan

Marintcheva

Cook

et al. 2005

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

150

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“…The E. coli SSB protein is required for the initiation of DNA synthesis (1), and it stimulates the loading of E. coli DNA primase (DnaG) onto DNA (52). E. coli SSB also participates in the transfer of newly synthesized primers to E. coli DNA polymerase III (39). We therefore examined how the T7 gp2.5 affects primer utilization by T7 DNA polymerase.…”

Section: Figmentioning

confidence: 99%

“…The proteinprotein interactions mediating the transfer of a primed template to DNA polymerase are starting to be elucidated in several different replication systems. During replication in E. coli, the primed DNA template is transferred from the DnaG primase to DNA polymerase III by the combined action of the ␤ clamp loader and the ssDNA-binding protein (SSB) (39). In contrast, T7 primase-helicase directly transfers a newly synthesized primer to T7 DNA polymerase and stimulates primer extension by the polymerase through an intimate physical interaction of the primase and polymerase (29,30,38).…”

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confidence: 99%

A Molecular Handoff between Bacteriophage T7 DNA Primase and T7 DNA Polymerase Initiates DNA Synthesis

Kato

Ito

Wagner

et al. 2004

Journal of Biological Chemistry

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The T7 DNA primase synthesizes tetraribonucleotides that prime DNA synthesis by T7 DNA polymerase but only on the condition that the primase stabilizes the primed DNA template in the polymerase active site. We used NMR experiments and alanine scanning mutagenesis to identify residues in the zinc binding domain of T7 primase that engage the primed DNA template to initiate DNA synthesis by T7 DNA polymerase. These residues cover one face of the zinc binding domain and include a number of aromatic amino acids that are conserved in bacteriophage primases. The phage T7 singlestranded DNA-binding protein gp2.5 specifically interfered with the utilization of tetraribonucleotide primers by interacting with T7 DNA polymerase and preventing a productive interaction with the primed template. We propose that the opposing effects of gp2.5 and T7 primase on the initiation of DNA synthesis reflect a sequence of mutually exclusive interactions that occur during the recycling of the polymerase on the lagging strand of the replication fork.DNA synthesis is initiated on the lagging strand of the replication fork by a site-specific RNA polymerase known as a DNA primase (1). The primase synthesizes a short RNA primer that is extended by DNA polymerase to create an Okazaki fragment (2, 3). During replication, interactions between the primase and other replicative proteins such as DNA polymerase, DNA helicase, and single-stranded DNA (ssDNA) 1 -binding protein are required to initiate DNA synthesis. It has been difficult to identify these essential interactions because they occur transiently and involve weak interactions between multiple partners.We have been studying the physical and functional interactions between proteins of the comparatively simple bacteriophage T7 replication system. Phage T7 replicates DNA using four proteins, T7 gene 4 primase-helicase protein (gp4), T7 gene 2.5 ssDNA-binding protein (gp2.5), and T7 DNA polymerase (gp5) complexed with its processivity factor, Escherichia coli thioredoxin (4). The reactions catalyzed by the individual proteins are coordinated during replication by the assembly of a multiprotein complex that moves with the replication fork (5, 6). During replication, the primase-helicase directly contacts both the DNA polymerase and gp2.5 (7-10). A C-terminal acidic segment of the primase-helicase is required for its interaction with the DNA polymerase (10). An interaction between gp2.5 and T7 DNA polymerase is required for the coordinated synthesis of both leading and lagging strands of the replication fork (5, 6). A C-terminal 21-residue region of gp2.5 is essential not only for the interactions with the DNA polymerase and the primase-helicase but also for dimerization of gp2.5 (9). Although many pairwise interactions between T7 replication proteins have been identified, the overall physical arrangement of subunits and their stoichiometries in the replication complex are unknown.Crystal structures of all the individual T7 replication proteins (11-16) have been determined, and their enzymatic ac...

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“…The ␥ complex (␥ 3 ␦␦Ј ) consists of a minimal core of five proteins (␥ 3 ␦␦Ј), which contain the clamp loading activity (11). The remaining two subunits, and , are involved in recruiting an RNA primed DNA site from the primase, and they bind singlestranded DNA-binding protein (SSB) to assist polymerase elongation but are not essential to the clamp loading activity of ␥ complex (12)(13)(14). Biochemical studies of ␥ complex (15)(16)(17), combined with crystal structures of ␥ 3 ␦␦Ј (10) and ␦-␤ 1 complex (18,19), reveal a highly detailed view of ␥ 3 ␦␦Ј clamp loader form and function.…”

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confidence: 99%

Replication Factor C Clamp Loader Subunit Arrangement within the Circular Pentamer and Its Attachment Points to Proliferating Cell Nuclear Antigen

Yao

Coryell

Zhang

et al. 2003

Journal of Biological Chemistry

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Replication factor C (RFC) is a heteropentameric AAA؉ protein clamp loader of the proliferating cell nuclear antigen (PCNA) processivity factor. The prokaryotic homologue, ␥ complex, is also a heteropentamer, and structural studies show the subunits are arranged in a circle. In this report, Saccharomyces cerevisiae RFC protomers are examined for their interaction with each other and PCNA. The data lead to a model of subunit order around the circle. A characteristic of AAA؉ oligomers is the use of bipartite ATP sites in which one subunit supplies a catalytic arginine residue for hydrolysis of ATP bound to the neighboring subunit. We find that the RFC(3/4) complex is a DNA-dependent ATPase, and we use this activity to determine that RFC3 supplies a catalytic arginine to the ATP site of RFC4. This information, combined with the subunit arrangement, defines the composition of the remaining ATP sites. Furthermore, the RFC(2/3) and RFC(3/4) subassemblies bind stably to PCNA, yet neither RFC2 nor RFC4 bind tightly to PCNA, indicating that RFC3 forms a strong contact point to PCNA. The RFC1 subunit also binds PCNA tightly, and we identify two hydrophobic residues in RFC1 that are important for this interaction. Therefore, at least two subunits in RFC make strong contacts with PCNA, unlike the Escherichia coli ␥ complex in which only one subunit makes strong contact with the ␤ clamp. Multiple strong contact points to PCNA may reflect the extra demands of loading the PCNA trimeric ring onto DNA compared with the dimeric ␤ ring.Replicases of cellular chromosomes utilize a circular sliding clamp protein that encircles DNA and tethers the polymerase to the template for high processivity in DNA synthesis (1). An example of this protein class is the Escherichia coli ␤ subunit, which confers high processivity onto the chromosomal replicase, DNA polymerase III holoenzyme (2, 3). The eukaryotic equivalent is the PCNA 1 ring, which has essentially the same shape and chain fold as ␤ despite lack of sequence similarity between the two (4, 5). These ring-shaped proteins require an ATP-fueled multiprotein clamp loader for assembly onto primed DNA.The eukaryotic clamp loader is the heteropentameric replication factor C (RFC). The five subunits of RFC are each different proteins, but they are homologous to one another (6, 7) and are members of the AAAϩ family of ATPases (8). The recent crystal structure of E. coli ␥ complex, the prokaryotic counterpart of RFC, has facilitated detailed hypothesis regarding RFC structure and mechanism (9, 10). The ␥ complex (␥ 3 ␦␦Ј ) consists of a minimal core of five proteins (␥ 3 ␦␦Ј), which contain the clamp loading activity (11). The remaining two subunits, and , are involved in recruiting an RNA primed DNA site from the primase, and they bind singlestranded DNA-binding protein (SSB) to assist polymerase elongation but are not essential to the clamp loading activity of ␥ complex (12)(13)(14). Biochemical studies of ␥ complex (15-17), combined with crystal structures of ␥ 3 ␦␦Ј (10) and ␦-␤ 1 complex ...

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Trading Places on DNA—A Three-Point Switch Underlies Primer Handoff from Primase to the Replicative DNA Polymerase

Cited by 211 publications

References 31 publications

A unique loop in T7 DNA polymerase mediates the binding of helicase-primase, DNA binding protein, and processivity factor

A unique loop in T7 DNA polymerase mediates the binding of helicase-primase, DNA binding protein, and processivity factor

A Molecular Handoff between Bacteriophage T7 DNA Primase and T7 DNA Polymerase Initiates DNA Synthesis

Replication Factor C Clamp Loader Subunit Arrangement within the Circular Pentamer and Its Attachment Points to Proliferating Cell Nuclear Antigen

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