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Gene 4 DNA Primase of Bacteriophage T7 Mediates the Annealing and Extension of Ribo-Oligonucleotides at Primase Recognition Sites

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Gene 4 DNA Primase of Bacteriophage T7 Mediates the Annealing and Extension of Ribo-Oligonucleotides at Primase Recognition Sites Gene 4 DNA Primase of Bacteriophage T7 Mediates the Annealing and Extension of Ribo-oligonucleotides at Primase Recognition Sites The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Kusakabe, Takahiro, and Charles C. Richardson. 1997. “Gene 4 DNA Primase of Bacteriophage T7 Mediates the Annealing and Extension of Ribo-Oligonucleotides at Primase Recognition Sites.” Journal of Biological Chemistry 272 (19): 12446–53. https://doi.org/10.1074/ jbc.272.19.12446. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41483409 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 19, Issue of May 9, pp. 12446–12453, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Gene 4 DNA Primase of Bacteriophage T7 Mediates the Annealing and Extension of Ribo-oligonucleotides at Primase Recognition Sites* (Received for publication, December 18, 1996) Takahiro Kusakabe and Charles C. Richardson‡ From the Department of Biological Chemistry and Molecular Pharmacology, Harvard University Medical School, Boston, Massachusetts 02115 The 63-kDa gene 4 primase of bacteriophage T7 recog- observed for the first two nucleotides polymerized at the basic nizes a core trinucleotide sequence, 5*-GTC-3*, on single- trinucleotide sequence (1, 3, 4, 7, 8, 11). stranded DNA at which it catalyzes the synthesis of the The molecular mechanism by which prokaryotic DNA pri- ribodinucleotide pppAC. The dinucleotide is extended mases recognize a trinucleotide sequence on ssDNA is not to a tetranucleotide primer at the sites 5*-(G/T)GGTC-3* known. However, the T7, T4, and E. coli primases have a and 5*-GTGTC-3*. In the presence of T7 primase, T7 DNA potential metal-binding site, as do all known DNA primases polymerase extends the synthetic ribotetranucleotide (12, 13); the T7 and E. coli primases have been shown to be zinc pACCA (1 mM), but not pCACA, on M13 DNA templates. metalloproteins (13, 14). In the case of the T7 primase, the Cys4 Downloaded from The reaction is specific for T7 DNA polymerase and de- zinc motif is located at the amino terminus of the protein; the pends on dTTP and translocation of the gene 4 protein. substitution of a serine for any one of the four cysteines de- T7 primase extends the dinucleotide AC and trinucle- stroys its ability to catalyze the synthesis of oligonucleotides in otide ACC to ACCC in the presence of CTP and an ap- propriate template, whereas other dinucleotides are ex- a sequence-specific manner (13). However, altered T7 primases tended less efficiently; the deoxyribodinucleotide dAC is containing these single amino acid changes or even lacking the entire zinc motif still catalyze the DNA-independent formation http://www.jbc.org/ not extended. The Cys4 zinc motif of the primase is es- sential for extension of the dinucleotides. The 5*-cryptic of random dinucleotides, albeit at a greatly reduced rate, dem- cytidine of the recognition sequence is essential for ex- onstrating that the site for phosphodiester bond formation is tension of the dinucleotide AC to tri- and tetranucleo- located in another domain of the protein (13, 15, 16). The zinc tides. At a preformed replication fork, the dinucleotide motif, however, is not the sole determinant of primase site AC provides for primer synthesis on the lagging strand. recognition since we have shown that substituting the zinc The synthesis of all Okazaki fragments is initiated by motifs of the T4 and E. coli primases for that of the T7 primase by guest on October 5, 2019 primers arising from the recognition sequence 5*- does not lead to the creation of a chimeric primase that now GGGTC-3*; none arise at an adjacent 5*-GGGTT-3* se- uses the T4 or E. coli recognition sequence, but rather to one quence. If ADP or AMP replaces ATP in the primase that recognizes an entirely new trinucleotide sequence (17). reaction, primers terminating in di- or monophosphate, Although the T7 primase shares many properties with the respectively, are synthesized. primases of phage T4 and E. coli, it has a number of distin- guishing properties. A unique feature of the T7 DNA primase is the presence of a helicase domain, which allows the 63-kDa DNA primases catalyze the template-directed synthesis of gene 4 protein, a protein composed of a single polypeptide oligonucleotides for use as primers by the lagging strand DNA chain, to catalyze both helicase and primase activities. (15, 18). polymerase. The primases of T7, T4, and Escherichia coli all In contrast, in the T4 and E. coli systems, helicase and primase initiate oligonucleotide synthesis on single-stranded DNA activities reside within separate polypeptides (8, 19–22). Phys- 1 (ssDNA) at basic trinucleotide recognition sites unique to each ical association of primases with a helicase in a functional system (1–5). The 39-nucleotide of the recognition sequence, in complex is important because the translocation activity of the each case, is cryptic in that it is essential for recognition, but is helicase provides a mechanism for the primase to reach its not copied into the product oligonucleotide. Although the basic recognition sites on ssDNA (2, 9, 20, 21, 23). The presence of recognition sequence is a trinucleotide, with the potential to both helicase and primase activities in the same T7 protein generate a dinucleotide, the oligonucleotides that actually func- arises from the presence of two co-linear gene 4 proteins, a 56- tion as primers for the T7, T4, and E. coli primases are tetra-, and a 63-kDa protein, in phage-infected cells (24). The two penta-, and 10–12-mers, respectively (6–10). The stringency proteins are expressed from two in-frame translation initiation for these subsequent nucleotide additions is less than that sites 189 bases apart on the gene 4 transcript. The 56-kDa gene 4 protein lacks the 63 N-terminal amino acid residues of the * This work was supported by United States Public Health Service 63-kDa gene 4 protein, and it is this domain that contains the Grant AI-06045 and by American Cancer Society Grant NP-1Z. The zinc motif essential for primase activity. Thus, the 56-kDa gene costs of publication of this article were defrayed in part by the payment 4 protein has only helicase activity, whereas the 63-kDa gene 4 of page charges. This article must therefore be hereby marked “adver- protein has both helicase and primase activities (16, 18). In this tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. report, we refer to the 63-kDa gene 4 protein as DNA primase ‡ To whom correspondence should be addressed: Dept. of Biological even though it has helicase activity. Chemistry and Molecular Pharmacology, Harvard University Medical A second distinguishing property of the T7 primase involves School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1864; Fax: the selectivity in the particular nucleotides incorporated into 617-432-3362; E-mail: [email protected]. 1 The abbreviations used are: ssDNA, single-stranded DNA; nt, the primers after the invariant dinucleotide is synthesized at nucleotide(s). the basic primase recognition site. T7 primase greatly prefers 12446 This paper is available on line at http://www-jbc.stanford.edu/jbc/ Ribodinucleotide Use by T7 Gene 4 Primase 12447 AMP and CMP in the third and fourth positions of the primer, CAAG AGATACAGTC GTTCGACAGA TGACATCCAG-39; T7-04, 59-T- whereas the T4 and E. coli enzymes are less restrictive in the TCTAGGACT ATCGGGCGAT CCCACAGTAG TAA-39; T7-05, 59-CCT- TCTCGCT GTGCCTTGTT TGCAGGTGCT TTAAAGATAC CACCA-3 ; nucleotides incorporated (25, 26). We have shown that this 9 MR-01, 59-TGGCGATTCG CAGTTTATAC CGATTCAGGT ACGTTAG- specificity is, at least in part, due to the inherent translocation GTA TCCATTGGTC TCCTAGGCTT AACGCCACGG-39; MR-04, 59-G- activity of the T7 primase, which does not allow the enzyme to GGTTCCAAG ACCTTAGGAT CCAGAGCCCA GCATTTGGAC TTAT- pause for sufficient time to incorporate unfavorable nucleotides GGGGGG GGGGGGGGGG GTCTCCGAAG CTTGGGGGG; TK-01, 59- (11). TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTATCGC TAACGT- A third distinguishing property, and the subject of this TACC TAGGTAGTAA GCTTAACCTC T-39; TK-02, 59-ATCGCTAACG- TTACCTAGGT AGTAAGCTTA ACCTCTACGT-3 ; and TK-03, 5 -AC- study, is the ability of the T7 primase to synthesize relatively 9 9 CTAGGTAA CGTTAGCGAT TACGGGATCC TCTCCGACCC GTTGG- large amounts of the dinucleotide pppAC together with the CAACC CACGTAGAGG TTAAGCTTAC T-39. The T7 63-kDa gene 4 functional primer species, a tetranucleotide. The actual RNA protein was purified as described (30). The T7 56-kDa gene 4 protein primers found at the 59-termini of Okazaki fragments synthe- was purified by B. Beauchamp (Harvard Medical School) as described sized in cells infected with phage T7 (6) or in reactions contain- (18). T7 gene 5 protein-E. coli thioredoxin (1:1 complex) was purified ing the T7 primase and T7 DNA polymerase are tetranucleo- and kindly provided by S. Tabor (Harvard Medical School) as described (31). We refer to the 1:1 complex of the gene 5 protein with thioredoxin tides, predominantly pppACCC, pppACCA, and pppACAC (7, as T7 DNA polymerase unless otherwise indicated. The T7 gene 2.5 26). These tetranucleotides arise from the general recognition ssDNA-binding protein was purified by D. Kong as described (32). T4 sites 59-GGGTC-39,59-TGGTC-39, and 59-GTGTC-39, respec- DNA polymerase, T4 polynucleotide kinase, T4 DNA ligase, and other tively, all containing the core recognition sequence for the T7 enzymes were purchased from Amersham Corp.
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