RNA-linked nascent DNA pieces in phage T7-infected Escherichia coli. II. Primary structure of the RNA portion

Sekit, Tetsunori; Okazaki, Tuneko

doi:10.1093/nar/7.6.1603

Cited by 38 publications

(23 citation statements)

References 22 publications

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“…4). ATP and CTP are the best pair of nucleotides for primase activity with either protein, a result consistent with the known T7 primase recognition sequence and the primers found at the terminus of Okazaki fragments both in vivo (32,33) and in vitro (5,16,17). Surprisingly, limited primase activity is also seen in the presence of ATP and GTP.…”

Section: Resultssupporting

confidence: 81%

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A functional chimeric DNA primase: the Cys4 zinc-binding domain of bacteriophage T3 primase fused to the helicase of bacteriophage T7.

Hine

Richardson

1994

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

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Two colinear bacteriophage T7 gene 4 proteins provide helicase and primase functions in vivo. T7 primase differs from 17 helicase by an additional 63 residues at the amino terminus. This terminal domain contains a zinc-binding motif which mediates an interaction with the basic primase recognition sequence 3'-CTG-5'. We have generated a chimeric primas in which the 81 amino-terminal residues are derived from the primase of phage T3 and the 484 carboxylterminal residues are those of phage 17 helicase. The aminoterminal domain of T3 primase is 50% homologous with that of T7 primase. The resulting T3/T7 chimeric protein is a functional primase in nvo. While the primase activity of the purified protein is about one-third that of T7 primase, the recognition sites used and the oligoribonucleotides synthesized from these sites are identical. We conclude that the residues responsible for the interaction with the sequence 3'-CTG-5' are conserved between the chimeric and 17 proteins.

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Section: Resultssupporting

confidence: 81%

“…4). This result was unexpected, since all characterized T7 primase recognition sites require the incorporation of CTP (5,33). Comparison of the lanes ACG and AG of Fig.…”

Section: Resultsmentioning

confidence: 97%

A functional chimeric DNA primase: the Cys4 zinc-binding domain of bacteriophage T3 primase fused to the helicase of bacteriophage T7.

Hine

Richardson

1994

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

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“…Not only were the amounts of dimer increased but the presence of tri-and tetranucleotide products were now observed. The latter are the only species that can serve as primers for T7 DNA polymerase either in vivo or in vitro (11,47). These results show that the chimeric proteins can still interact physically with the 56-kDa helicase, presumably to form hexamers and to provide translocation activity for the chimeric primase in the complex.…”

Section: Generation Of Chimeric Primases-mentioning

confidence: 97%

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

Kusakabe

Richardson

1996

Journal of Biological Chemistry

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The DNA primase of bacteriophage T7 has a zinc-binding motif that is essential for the recognition of the sequence 3-CTG-5. The T7 primase also catalyzes helicase activity, a reaction coupled to nucleotide hydrolysis. We have replaced the zinc motif of the T7 primase with those found in the gene 61 primase of phage T4 and the DnaG primase of Escherichia coli. The T4 and E. coli primases recognize the sequences 3-T(C/T)G-5 and 3-GTC-5, respectively. Both chimeric proteins can partially replace T7 primase in vivo. The two chimeric primases catalyze the synthesis of oligoribonucleotides albeit at a reduced rate and DNA dependent dTTPase activity is reduced by 3-10-fold. Both chimeric proteins recognize 3-(A/G)CG-5 sites on single-stranded DNA, sites that differ from those recognized by the T7, T4, or E. coli primases, indicating that the zinc motif is only one determinant in site-specific recognition.

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“…2 The findin class I sites in segments II and III were on or before the were detectc TTAA sequence, which occurs frequently in the primary conditions si origin region. Unexpectedly, six sites (sites 2, 8, 10, 13, 15, RNApolym and 18) fell to the gene 4 primase sites and at another six sites primers for (sites 3, 6, 17, 19, 20, and 21), ACC or CCC sequences (typical replication f sequences found in the T7 phage primer RNA) were found sites in the g (36). The class II transition sites (indicated by uppercase remains thai letters in Fig.…”

Section: Methodsmentioning

confidence: 95%

Relative roles of T7 RNA polymerase and gene 4 primase for the initiation of T7 phage DNA replication in vivo.

Sugimoto¹,

Kohara²,

Okazaki³

1987

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

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Initiation sites of T7 phage DNA replication in the presence and absence of T7 phage gene 4 primase have been analyzed by using Escherichia coli cells infected with T7 phage amber mutants, T73,6 and T73,4,6, respectively. Restriction analysis of the [3H]thymidine-labeled DNA, synthesized by the T73,4,6 phage-infected cells in the presence of 2',3'-dideoxy-3'-azidothymidine, has shown that only the light (L) strand of T7 DNA has been synthesized from the primary origin area to the right. Transition sites from RNA to DNA have been located precisely in the primary origin region of the T7 phage genome. In the gene 4-condition, >20 transition sites have been detected only in the L strand. They scattered widely downstream from the 4b1.1 promoters and mostly downstream from the 41.3 promoter. The same transition sites have been detected in the gene 4+ condition, suggesting that the transcripts started from these promoters are used as primers of the rightward L-strand DNA synthesis in the gene 4+ condition. In addition, many heavy (H)-and L-strand transition sites have been detected at gene 4 primase sites in the gene 4+ condition. The relative roles of T7 phage RNA polymerase and primase at the primary origin have been discussed.In many prokaryotic organisms, including bacteriophage T7, transcription by RNA polymerase is directly required for the initiation of DNA replication (1, 2). Two possibilities exist for the required function(s): one is activation of the replication origin (3) and the other is priming of the DNA synthesis. The enzyme might exert dual functions. Alternatively, the first primer at the replication origin is made by DNA primase and the transcriptional activation is prerequisite to it. The purpose of this study is to assess the primer function of the transcript by T7 phage RNA polymerase and primase (gene 4 protein) in the initiation of the T7 phage DNA replication.The bacteriophage T7 chromosome (40 kilobase pairs, linear duplex) replicates as a linear monomer at an early stage of infection (4). The replication first creates an eye structure at 17 map units on the phage genome and proceeds bidirectionally (4). A 126-base-pair (bp) intergenic segment from 14.75 to 15.0 map units, called the primary origin, is required in cis for the eye formation (5, 6). Concatemers are formed in the subsequent rounds of replication, in which different origin(s) might function (7).The T7 phage primary origin contains two T7 RNA polymerase promoters (41.1A and 41.1B) and a 61-bp (A+T)-rich (78% A+T) region in which gene 4 primase sites are contained (see Fig. 4 RNA polymerase from either one of the 01.1 promoters is used as a primer for the L-strand DNA synthesis. The actual priming mechanism could be examined by mapping exact transition sites from primer RNA to DNA, since recognized signal sequences and transcripts of 17 RNA polymerase and of gene 4 primase are quite distinctive from each other: the promoters of T7 RNA polymerase are 23-bp well-conserved stretches and transcripts are exclusively composed of t...

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RNA-linked nascent DNA pieces in phage T7-infected Escherichia coli. II. Primary structure of the RNA portion

Cited by 38 publications

References 22 publications

A functional chimeric DNA primase: the Cys4 zinc-binding domain of bacteriophage T3 primase fused to the helicase of bacteriophage T7.

A functional chimeric DNA primase: the Cys4 zinc-binding domain of bacteriophage T3 primase fused to the helicase of bacteriophage T7.

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

Relative roles of T7 RNA polymerase and gene 4 primase for the initiation of T7 phage DNA replication in vivo.

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