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A Unique Loop in the DNA-Binding Crevice of Bacteriophage T7 DNA Polymerase Influences Primer Utilization

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A Unique Loop in the DNA-Binding Crevice of Bacteriophage T7 DNA Polymerase Influences Primer Utilization A unique loop in the DNA-binding crevice of bacteriophage T7 DNA polymerase influences primer utilization The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Chowdhury, K., S. Tabor, and C. C. Richardson. 2000. “A Unique Loop in the DNA-Binding Crevice of Bacteriophage T7 DNA Polymerase Influences Primer Utilization.” Proceedings of the National Academy of Sciences 97 (23): 12469–74. https://doi.org/10.1073/ pnas.230448397. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41483329 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 A unique loop in the DNA-binding crevice of bacteriophage T7 DNA polymerase influences primer utilization Kajal Chowdhury, Stanley Tabor, and Charles C. Richardson* Department of Biological Chemistry and Molecular Pharmacology, Harvard University Medical School, Boston, MA 02115 Contributed by Charles C. Richardson, September 19, 2000 The three-dimensional structure of bacteriophage T7 DNA poly- The 63-kDa gene 4 protein contains a Cys4 zinc-binding motif merase reveals the presence of a loop of 4 aa (residues 401–404) (26, 27) within the additional 63 aa at its N terminus not present within the DNA-binding groove; this loop is not present in other in the 56-kDa species. In addition to having helicase activity, the members of the DNA polymerase I family. A genetically altered T7 63-kDa gene 4 protein acts as a primase to catalyze the synthesis DNA polymerase, T7 pol⌬401–404, lacking these residues, has been of template-directed oligoribonucleotides that serve as primers characterized biochemically. The polymerase activity of T7 for T7 DNA polymerase (9, 19, 26, 28, 29). Gene 4 primase pol⌬401–404 on primed M13 single-stranded DNA template is synthesizes functional tetraribonucleotide primers 5Ј-pp- one-third of the wild-type enzyme and has a 3؅-to-5؅ exonuclease pACCC-3Ј,5Ј-pppACCA-3Ј, and 5Ј-pppACAC-3Ј at primase activity indistinguishable from that of wild-type T7 DNA polymer- recognition sequences 5Ј-GGGTC-3Ј,5Ј-TGGTC-3Ј, and 5Ј- ase. T7 pol⌬401–404 polymerizes nucleotides processively on a GTGTC-3Ј, respectively (30–33). All of these primase recogni- primed M13 single-stranded DNA template. T7 DNA polymerase tion sequences contain the core recognition sequence 5Ј-GTC- cannot initiate de novo DNA synthesis; it requires tetraribonucle- 3Ј. Interestingly, the 3Ј-cytidine in the primase recognition otides synthesized by the primase activity of the T7 gene 4 protein sequence is required but not copied into the primer. The to serve as primers. T7 primase-dependent DNA synthesis on utilization of the tetraribonucleotides as primers by T7 DNA single-stranded DNA is 3- to 6-fold less with T7 pol⌬401–404 polymerase requires the presence of T7 gene 4 protein; other compared with the wild-type enzyme. Furthermore, the altered primases will not function in this reaction (29, 32). One role of polymerase is defective (10-fold) in its ability to use preformed the gene 4 protein in this reaction is to stabilize the tetraribo- tetraribonucleotides to initiate DNA synthesis in the presence of nucleotide onto the template until it can be extended by the gene 4 protein. The location of the loop places it in precisely the polymerase (32). In addition, the specific requirement for gene position to interact with the tetraribonucleotide primer and, pre- 4 helicase͞primase may reflect an interaction between the sumably, with the T7 gene 4 primase. Gene 4 protein also provides primase domain of the gene 4 protein and T7 DNA polymerase helicase activity for the replication of duplex DNA. T7 pol⌬401–404 for the primer to be transferred to the active site of the and T7 gene 4 protein catalyze strand-displacement DNA synthesis polymerase. In addition to the postulated interactions of the at nearly the same rate as does wild-type polymerase and T7 gene primase with the polymerase, it is likely that the helicase domain 4 protein, suggesting that the coupling of helicase and polymerase of the gene 4 protein also interacts with the polymerase. A activities is unaffected. carboxyl-terminal deletion mutant of gene 4 protein that retains a normal level of helicase and primase activities fails to support pecific protein–protein interactions are essential for the strand displacement DNA synthesis by T7 DNA polymerase Scoordination of leading and lagging strand-DNA synthesis at (25). Furthermore, an N-terminal fragment of T7 gene 4 protein a replication fork. The relatively few proteins required for retaining only primase activity is unable to prime DNA synthesis coordinated DNA synthesis in the bacteriophage T7 replication catalyzed by T7 DNA polymerase (34), indicating the impor- BIOCHEMISTRY system make it an attractive model for studying these interactions tance of the helicase domain in the proper utilization of the (1). Only four proteins are required for the basic steps at the tetraribonucleotide primer by T7 DNA polymerase. replication fork: T7 gene 5 DNA polymerase, gene 4 helicase͞ Structure–function studies of T7 DNA polymerase have been primase, gene 2.5 single-stranded DNA (ssDNA)-binding pro- facilitated by the solution of its crystal structure (35). One tein, and Escherichia coli thioredoxin (1, 2). T7 gene 5 DNA approach to identify domains of T7 DNA polymerase that polymerase by itself has low processivity of DNA synthesis and interact with other T7 replication proteins is to identify regions requires E. coli thioredoxin in a 1:1 complex to achieve high in the proteins that are unique among other members of the processivity (3–7). In this communication we will refer to this DNA polymerase I (pol I) family. This approach revealed the functional 1:1 complex of T7 gene 5 protein and thioredoxin as presence of a loop of four residues (Glu-Gly-Asp-Lys) spanning T7 DNA polymerase. T7 gene 2.5 ssDNA-binding protein in- residues 401–404. Such a loop is absent in the other members of ͞ the pol I family whose structures are known (35–38). Interest- teracts with T7 gene 4 helicase primase (8–10) and also with T7 Ј DNA polymerase through its acidic carboxyl-terminal domain ingly, this loop is 4 bases away from 3 -OH terminus of the (10, 11). primer. Because T7 primase stabilizes the tetraribonucleotide T7 gene 4 protein exists in two forms with molecular weights primer (32), this location of the loop likely places it in close of 56,000 and 63,000. The 56-kDa species is translated from an proximity to the primase domain of gene 4 protein. internal initiation codon, in-frame with the 63-kDa species (12–15). Both forms of gene 4 protein bind to ssDNA as a Abbreviations: pol I, DNA polymerase I; ssDNA, single-stranded DNA; dsDNA, double- hexamer (16–18) in the presence of nucleoside 5Ј-triphosphates, Ј Ј stranded DNA. preferentially dTTP, and translocate 5 to 3 by using the energy *To whom reprint request should be addressed. E-mail: [email protected]. of nucleotide hydrolysis (13, 19–21). Upon encountering duplex Ј The publication costs of this article were defrayed in part by page charge payment. This DNA with six to seven unpaired nucleotides at the 3 end, the article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. gene 4 protein unwinds the DNA strands processively (20, §1734 solely to indicate this fact. 22–24). The acidic carboxyl terminus of T7 gene 4 protein Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.230448397. interacts with T7 DNA polymerase (25). Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.230448397 PNAS ͉ November 7, 2000 ͉ vol. 97 ͉ no. 23 ͉ 12469–12474 To assess the role of the loop in interactions of T7 DNA DNA polymerase. The reactions were initiated by the addition polymerase with gene 4 helicase͞primase and with DNA, we of the enzyme and incubated at 37°C for the indicated time have constructed a plasmid encoding for T7 gene 5 lacking the periods. The reaction was terminated by the addition of 25 ␮lof four-residue loop. We have purified and characterized the 0.5 mM EDTA. The [32P]DNA remaining was adsorbed onto altered T7 DNA polymerase lacking the loop (T7 pol⌬401–404). DE81 filter paper and washed three times with 0.3 M ammon- Our results indicate that this four-residue loop indeed is impor- ium formate (pH 8.0), and the radioactivity was measured by tant for interacting with the T7 gene 4 primase–tetraribonucle- liquid scintillation counting to calculate the amount of DNA otide primer complex. hydrolyzed. Materials and Methods T7 Gene 4-Dependent DNA Synthesis by T7 DNA Polymerase. The Mutagenesis of T7 Gene 5. In vitro mutagenesis of bacteriophage T7 ability of T7 DNA polymerase to extend a tetraribonucleotide in gene 5 to delete the region encoding amino acids 401–404 was the presence of gene 4 primase was assayed by using two accomplished by standard PCR techniques by using two syn- different methods. In one case, T7 63-kDa gene 4 protein was thetic primers that encode the deletion and the plasmid pGP5–3 allowed to synthesize the tetraribonucleotide primers at the (7) as a DNA template. After amplification, the resulting major primase recognition sites on a circular M13 ssDNA amplified DNA fragment was digested with restriction enzymes template in the presence of ATP and CTP as described (9, 32), and inserted into pGP5–3 to generate the plasmid pGP5–3⌬401– and the ability of T7 DNA polymerase to extend the tetraribo- 404.
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