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J. Biol. Chem., Vol. 281, Issue 35, 25635-25643, September 1, 2006

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Mechanistic Coupling of Bacteriophage T4 DNA Packaging to Components of the Replication-dependent Late Transcription Machinery^*

From the Department of Biochemistry and Molecular Biology, University of Maryland Medical School, Baltimore, Maryland 21201-1503

Regulation of the terminal stage of viral DNA development, DNA packaging, is poorly understood. A new phage T4 in vitro DNA packaging assay employed purified proheads, terminase (gp17 + gp16), and ATP to encapsidate DNA resistant to nuclease. Mature phage T4 DNA and linearized plasmid DNAs containing or lacking a cloned T4 gene were packaged with high (10%) efficiency. Supercoiled, relaxed covalently closed, and nicked circular plasmid DNAs were packaged inefficiently, if at all, by these components. However, efficient packaging is achieved for nicked circular plasmid DNA, but not covalently closed plasmid DNA, upon addition to packaging mixtures of the purified T4 late transcription-replication machinery proteins: gp45 (sliding clamp), gp44/gp62 (clamp loader complex), gp55 (late -factor), and gp33 (transcriptional co-activator). The small terminase subunit (gp16) is inhibitory for packaging linear DNAs, but enhances the transcription-replication protein packaging of nicked plasmid DNA. Taken together with genetic and biochemical evidence of a requirement for gp55 for concatemer packaging to assemble active wild-type phage particles (1), the plasmid packaging results show that initiation of phage T4 packaging on "endless" concatemeric DNA in vivo by terminase depends upon interaction with the DNA loaded gp45 coupled late transcription-replication machinery. The results suggest a close mechanistic connection in vivo between DNA packaging and developmentally concurrent replication-dependent late transcription.

Received for publication, March 6, 2006 , and in revised form, June 5, 2006.

^* This work was supported by National Institutes of Health Grant AI11676 (to L. W. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Maryland Medical School, Rm 408, 108 N. Greene St., Baltimore, MD 21201-1503. Tel.: 410-706-3510; Fax: 410-706-8297; E-mail: lblack{at}umaryland.edu.

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