Single-stranded DNA binding protein and DNA helicase of bacteriophage T7 mediate homologous DNA strand exchange.

Kong, Daochun; Richardson, Charles C.

doi:10.1002/j.1460-2075.1996.tb00552.x

Cited by 60 publications

(69 citation statements)

References 57 publications

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“…In addition to mere RNA replication, nsp9 could also participate in such a base-pairing-driven process as RNA processing. An informative parallel in the virus world is observed with bacteriophage T7: its gene 2.5 ssDNA-binding protein binds substrates with similar affinity as SARS-CoV nsp9 does [k d in the M range (37)] and is involved in replication͞recombination͞homologous basepairing events (38,39). The structural and functional characterization of nsp9 may also be relevant to SARS-CoV control: the SARS epidemics as well as previous work on CoVs have shown that genome plasticity (evolution by mutation and recombination) relate to pathogenicity and probably also to drug resistance.…”

Section: Resultsmentioning

confidence: 99%

The severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded RNA-binding subunit unique in the RNA virus world

Egloff

Ferrón

Campanacci

et al. 2004

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

280

328

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The recently identified etiological agent of the severe acute respiratory syndrome (SARS) belongs to Coronaviridae (CoV), a family of viruses replicating by a poorly understood mechanism. Here, we report the crystal structure at 2.7-Å resolution of nsp9, a hitherto uncharacterized subunit of the SARS-CoV replicative polyproteins. We show that SARS-CoV nsp9 is a single-stranded RNA-binding protein displaying a previously unreported, oligosaccharide͞oligo-nucleotide fold-like fold. The presence of this type of protein has not been detected in the replicative complexes of RNA viruses, and its presence may reflect the unique and complex CoV viral replication͞transcription machinery.I n 2003, a human coronavirus (CoV) was identified as the causative agent of a form of atypical pneumonia: severe acute respiratory syndrome-CoV (SARS-CoV) (1-5). Coronaviridae have the longest known single-stranded (ss)RNA genome (27-31.5 kb), with a complex genetic organization and sophisticated replication͞transcription cycle (6, 7). Twenty-eight proteins are predicted to be encoded by the SARS-CoV genome (8, 9). The nonstructural (nsp) or ''replicase'' proteins of CoVs are derived from an unusually large replicase gene of Ͼ20 kb that consists of two large ORFs (ORFs 1a and 1b). Translation of this replicase gene from the incoming genomic RNA is the first step in CoV genome expression and includes a Ϫ1 ribosomal frameshift to express the ORF1b-encoded polypeptide. Translation products are the pp1a polyprotein (Ͼ4,000 amino acids) and the C-terminally extended pp1ab polyprotein (Ͼ7,000 amino acids), which are both cleaved by two or three ORF1a-encoded viral proteinases (10). Most of these replicase cleavage products assemble into a membrane-associated viral replication͞ transcription complex. Among other components, this complex includes a set of relatively small polypeptides (nsp6 to nsp11) encoded by the 3Ј region of ORF1a, for which no predicted nor proven function has been assigned. For the mouse hepatitis CoV, several of these cleavage products were reported to colocalize with other components of the viral replication complex in the perinuclear region of the infected cell (11), suggesting their involvement (directly or indirectly) in viral RNA metabolism.As part of a viral structural genomics program (12), we have cloned the 28 gene products of SARS-CoV and expressed them either as full-length proteins or as (predicted) functional domains. The determination of the three-dimensional structures of these gene products is expected to facilitate and accelerate discovery of drugs against this emerging and life-threatening pathogen. Furthermore, structural homology search is becoming a powerful method to infer biochemical and͞or biological function of previously uncharacterized proteins. We report here the crystal structure of nsp9, one the SARS-CoV uncharacterized nonstructural protein, as well as evidence for its function as an ssDNA͞RNA-binding protein. Materials and MethodsCrystallization, Structure Determination, and Refinement. SARS...

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Section: Resultsmentioning

confidence: 99%

The severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded RNA-binding subunit unique in the RNA virus world

Egloff

Ferrón

Campanacci

et al. 2004

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

280

328

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“…The 63-kDa T7 gene 4 protein, a species of gene 4 protein that has both helicase and primase activities, was overexpressed and purified to apparent homogeneity (Ͼ98% pure) from E. coli cells by B. Beauchamp (Harvard Medical School) as described (22). T7 gene 2.5 protein was purified to apparent homogeneity (Ͼ98% pure) as previously described (2). T7 gene 6 protein (5Ј-to-3Ј exonuclease), E. coli single-stranded DNA-binding protein (SSB protein) and restriction enzymes were purchased from Amersham.…”

Section: Methodsmentioning

confidence: 99%

“…Consequently, we believe that the T7 gene 4 helicase-mediated strand exchange reaction provides the major pathway for recombination in E. coli cells infected with bacteriophage T7 (2). In the present study we have examined the ability of the T7 gene 4 helicase to participate in strand transfer reactions to produce heteroduplex molecules containing pyrimidine dimers, base pair mismatches, and insertions in each of the two DNA strands similar to those described above for the E. coli RecA protein.…”

mentioning

confidence: 93%

“…1). In the preparation of this joint molecule we have used the T7 gene 2.5 ssDNA-binding protein to mediate the efficient annealing of the two partner molecules (2,3). As depicted in Fig.…”

mentioning

confidence: 99%

“…As depicted in Fig. 1, strand transfer driven by the helicases is coupled to the hydrolysis of a nucleoside 5Ј-triphosphate and proceeds in a polar 5Ј-to-3Ј direction with respect to the invading strand (2,3).…”

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Gene 4 helicase of bacteriophage T7 mediates strand transfer through pyrimidine dimers, mismatches, and nonhomologous regions

Kong

Griffith

Richardson

1997

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

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In bacteriophage T7 the gene 2.5 singlestranded DNA-binding protein and the gene 4 helicase together promote the annealing of homologous regions of two DNA partners to form a joint molecule and subsequent strand transfer. In this reaction T7 gene 2.5 protein is essential for joint molecule formation, but is not required for T7 gene 4 protein-mediated strand transfer. T7 gene 4 helicase alone is able to mediate strand transfer, provided that a joint molecule is available. The present paper shows that, in addition, strand transfer proceeds at a normal rate even when both DNA partners contain ultraviolet-induced pyrimidine dimers (0.6 dimer per 100 nt). An insert of a relatively long (842-nt) segment of nonhomologous DNA in the single-stranded DNA partner has no effect on strand transfer, whereas its presence in the double-stranded partner prevents strand transfer. A short insert (37 nt) can be tolerated in either partner. Thus, DNA helicase is able to participate in recombinational DNA repair through its role in strand exchange, providing a pathway distinct from nucleotide excision repair.Recent studies have shown that the gene 41 DNA helicase encoded by bacteriophage T4 (1) and the gene 4 helicase encoded by bacteriophage T7 (2) mediate homologous DNA strand transfer within homologous DNA molecules. The rate and polarity of strand transfer are in accord with the properties of these enzymes in catalyzing strand displacement at a replication fork. A convenient DNA substrate for dissecting the strand transfer reaction is a joint DNA molecule formed between a single-stranded circle and a homologous linear duplex DNA molecule with a short 3Ј-single-stranded tail (Fig.

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T 7 Bacteriophages

Molineux¹

2002

Encyclopedia of Molecular Biology

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Single-stranded DNA binding protein and DNA helicase of bacteriophage T7 mediate homologous DNA strand exchange.

Cited by 60 publications

References 57 publications

The severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded RNA-binding subunit unique in the RNA virus world

The severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded RNA-binding subunit unique in the RNA virus world

Gene 4 helicase of bacteriophage T7 mediates strand transfer through pyrimidine dimers, mismatches, and nonhomologous regions

T 7 Bacteriophages

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