The Role of the Zinc Motif in Sequence Recognition by DNA Primases

Kusakabe, Takahiro; Richardson, Charles C.

doi:10.1074/jbc.271.32.19563

Cited by 58 publications

(57 citation statements)

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“…The 3Ј-cytidine is required for recognition but is not copied into the primer. Although limited synthesis of pppAA dinucleotide has been observed to occur at the trinucleotide sequence 5Ј-TTC-3Ј under certain conditions, the pppAA dinucleotide is not extended further (6,21). Analysis of the oligoribonucleotide primers attached to the 5Ј-end of Okazaki fragments both in vivo and in vitro has shown that the T7 primase synthesizes predominantly pppAC(C/A)(C/A) tetraribonucleotides; UMP and GMP are not found in the primers (17,18,20).…”

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“…Although the zincbinding motif is essential for primer synthesis (5), it alone cannot account for the ability of the protein to recognize specific sequences on ssDNA (6). Gene 4 protein is essential for phage growth and physically interacts with both the gene 5 DNA polymerase and the gene 2.5 ssDNA binding protein to mediate leading and lagging strand synthesis at the T7 replication fork (7)(8)(9)(10)(11)(12).…”

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Template Recognition and Ribonucleotide Specificity of the DNA Primase of Bacteriophage T7

Kusakabe

Richardson

1997

Journal of Biological Chemistry

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The 63-kDa gene 4 DNA primase of phage T7 catalyzes the synthesis of oligoribonucleotides on single-stranded DNA templates. At the sequence, 5-GTC-3, the primase synthesizes the dinucleotide pppAC; the cytidine residue of the recognition sequence is cryptic. Only tetraribonucleotides function as primers, but the specificity for the third and fourth position is not as stringent with a preference of CMP > AMP > > UMP > GMP. The predominant recognition sites on M13 DNA are 5-(G/T)G-GTC-3 and 5-GTGTC-3. Synthesis is usually limited to tetranucleotides, but T7 primase can synthesize longer oligoribonucleotides on templates containing long stretches of guanosine residues 5 to the recognition sequence. The specificity beyond the first two positions of the primer increases as the length of the template on the 3-side of 5-GTC-3 increases. On an oligonucleotide having 20 3-flanking cytidine residues GMP is incorporated at the third position; incorporation is reduced 4-fold when the flanking sequence reaches 65 residues, and little is incorporated on M13 templates. The presence of the 56-kDa gene 4 helicase decreases the incorporation of GMP on long templates. We propose that pausing is required for the incorporation of less preferred nucleotides and that pausing is decreased by the ability of the primase to translocate 5 to 3 on templates having long 3-flanking sequences.

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Template Recognition and Ribonucleotide Specificity of the DNA Primase of Bacteriophage T7

Kusakabe

Richardson

1997

Journal of Biological Chemistry

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“…DnaG-type primase structures exhibit zinc-ribbon domains (15,26,27) that are tethered loosely to their catalytic domains and regulate initiation of primer synthesis and length (26)(27)(28)(29). Pri-type primases feature zincknuckle or zinc-stem structures inserted in their PriS-catalytic domain that were suggested to enforce the interaction with the ssDNA template, but were shown not to be essential for primase activity, in contrast to the zinc ribbon domain (18,20).…”

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Structure and function of primase RepB′ encoded by broad-host-range plasmid RSF1010 that replicates exclusively in leading-strand mode

Geibel

Banchenko

Engel

et al. 2009

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

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For the initiation of DNA replication, dsDNA is unwound by helicases. Primases then recognize specific sequences on the template DNA strands and synthesize complementary oligonucleotide primers that are elongated by DNA polymerases in leading-and lagging-strand mode. The bacterial plasmid RSF1010 provides a model for the initiation of DNA replication, because it encodes the smallest known primase RepB (35.9 kDa), features only 1 single-stranded primase initiation site on each strand (ssiA and ssiB, each 40 nt long with 5 -and 3 -terminal 6 and 13 single-stranded nucleotides, respectively, and nucleotides 7-27 forming a hairpin), and is replicated exclusively in leading strand mode. We present the crystal structure of full-length dumbbell-shaped RepB consisting of an N-terminal catalytic domain separated by a long ␣-helix and tether from the C-terminal helixbundle domain and the structure of the catalytic domain in a specific complex with the 6 5 -terminal single-stranded nucleotides and the C7-G27 base pair of ssiA, its single-stranded 3 -terminus being deleted. The catalytic domains of RepB and the archaeal/eukaryotic family of Pri-type primases share a common fold with conserved catalytic amino acids, but RepB lacks the zinc-binding motif typical of the Pri-type primases. According to complementation studies the catalytic domain shows primase activity only in the presence of the helix-bundle domain. Primases that are highly homologous to RepB are encoded by broad-host-range IncQ and IncQ-like plasmids that share primase initiation sites ssiA and ssiB and high sequence identity with RSF1010.Pri-type ͉ primase DNA complex ͉ single-strand initiator DNA ͉ crystal structure ͉ oriV A t the initiation of DNA replication, the double helix is unwound by helicases, and then primases synthesize complementary oligonucleotide primers on the single-stranded template DNA. These primers are extended by DNA polymerase, continuously at the leading strand and discontinuously at the oppositely-oriented lagging strand, where Okazaki fragments are synthesized. Primase activity depends on specific ssDNA priming sequences that are periodically required for the initiation of the Okazaki fragments on the lagging strand (that are connected by ligase) but only 1 primer is used for the leading strand (1).The broad-host-range IncQ plasmid RSF1010 is maintained in Ͼ30 Gram-negative and 2 Gram-positive bacteria (2, 3). The replication of IncQ and IncQ-like plasmids and their conjugative transfer was studied in Escherichia coli and in vitro. RSF1010 encodes helicase RepA, primase RepBЈ, and replication initiator protein RepC (4, 5) (Fig. S1 A). Additionally, the replication of RSF1010 requires bacterial host replication proteins such as DNA polymerase III and single-stranded binding protein.Primase RepBЈ is activated on 2 specific, 40-nt-long singlestranded segments termed initiators A and B (ssiA and ssiB) with highly similar nucleotide sequences (6, 7). They are arranged on opposite DNA strands as a palindrome in the origin of vegetative re...

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“…1A for T7 DNA primase) contain six conserved sequence motifs (9). An N-terminal zinc-binding domain (ZBD) formed by motif I is a determinant for recognition of a specific sequence in DNA (10)(11)(12)(13). Motifs II-VI are located in the C-terminal RNA polymerase domain (RPD) that consists of two subdomains (Fig.…”

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Direct role for the RNA polymerase domain of T7 primase in primer delivery

Zhu

Lee²,

Richardson³

2010

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

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Gene 4 protein (gp4) encoded by bacteriophage T7 contains a C-terminal helicase and an N-terminal primase domain. After synthesis of tetraribonucleotides, gp4 must transfer them to the polymerase for use as primers to initiate DNA synthesis. In vivo gp4 exists in two molecular weight forms, a 56-kDa form and the full-length 63-kDa form. The 56-kDa gp4 lacks the N-terminal Cys 4 zinc-binding motif important in the recognition of primase sites in DNA. The 56-kDa gp4 is defective in primer synthesis but delivers a wider range of primers to initiate DNA synthesis compared to the 63-kDa gp4. Suppressors exist that enable the 56-kDa gp4 to support the growth of T7 phage lacking gene 4 (T7Δ4). We have identified 56-kDa DNA primases defective in primer delivery by screening for their ability to support growth of T7Δ4 phage in the presence of this suppressor. Trp69 is critical for primer delivery. Replacement of Trp69 with lysine in either the 56-or 63-kDa gp4 results in defective primer delivery with other functions unaffected. DNA primase harboring lysine at position 69 fails to stabilize the primer on DNA. Thus, a primase subdomain not directly involved in primer synthesis is involved in primer delivery. The stabilization of the primer by DNA primase is necessary for DNA polymerase to initiate synthesis.T7 bacteriophage | primer handoff D NA polymerases cannot initiate synthesis of DNA de novo, requiring a 3′-hydroxyl terminated primer positioned at the catalytic site. During DNA replication, primers are synthesized by a class of enzymes designated DNA primases (1). DNA primases catalyze the synthesis of short oligoribonucleotides that can then be used as primers by DNA polymerase. DNA primases play roles in the loading of DNA helicase (2), temporal regulation of replication (3-5), and handoff of the primer to the DNA polymerase (6-8).Based on their sequences and structures, DNA primases can be grouped into two families-the prokaryotic and the eukaryotic/ archaeal primases (1). Prokaryotic primases (see Fig. 1A for T7 DNA primase) contain six conserved sequence motifs (9). An N-terminal zinc-binding domain (ZBD) formed by motif I is a determinant for recognition of a specific sequence in DNA (10-13). Motifs II-VI are located in the C-terminal RNA polymerase domain (RPD) that consists of two subdomains (Fig. 1A). The C-terminal topoisomerase-primase (TOPRIM) fold (14) contains the active site where the metal-mediated condensation of nucleotides occurs. The N-terminal subdomain of the RPD is less conserved (6), and its role remains elusive. A recent study suggests that this subdomain contains a ssDNA binding groove through which the template can track (15).Gene 4 of bacteriophage T7 encodes two colinear proteins. The full-length product is a 63-kDa protein (63-kDa gp4) in which the C-terminal half encodes the helicase and the N-terminal half the primase (Fig. 1B). The coexistence of both primase and helicase in a single polypeptide is unique in that the two activities are physically associated in other systems but are e...

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The Role of the Zinc Motif in Sequence Recognition by DNA Primases

Cited by 58 publications

References 58 publications

Template Recognition and Ribonucleotide Specificity of the DNA Primase of Bacteriophage T7

Template Recognition and Ribonucleotide Specificity of the DNA Primase of Bacteriophage T7

Structure and function of primase RepB′ encoded by broad-host-range plasmid RSF1010 that replicates exclusively in leading-strand mode

Direct role for the RNA polymerase domain of T7 primase in primer delivery

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