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J. Biol. Chem., Vol. 281, Issue 1, 518-527, January 6, 2006

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Functional Analysis of the Bacteriophage T4 DNA-packaging ATPase Motor^*

From the Department of Biology, The Catholic University of America, Washington, D. C. 20064

Packaging of double-stranded DNA into bacteriophage capsids is driven by one of the most powerful force-generating motors reported to date. The phage T4 motor is constituted by gene product 16 (gp16) (18 kDa; small terminase), gp17 (70 kDa; large terminase), and gp20 (61 kDa; dodecameric portal). Extensive sequence alignments revealed that numerous phage and viral large terminases encode a common Walker-B motif in the N-terminal ATPase domain. The gp17 motif consists of a highly conserved aspartate (Asp²⁵⁵) preceded by four hydrophobic residues (²⁵¹MIYI²⁵⁴), which are predicted to form a -strand. Combinatorial mutagenesis demonstrated that mutations that compromised hydrophobicity, or integrity of the -strand, resulted in a null phenotype, whereas certain changes in hydrophobicity resulted in cs/ts phenotypes. No substitutions, including a highly conservative glutamate, are tolerated at the conserved aspartate. Biochemical analyses revealed that the Asp²⁵⁵ mutants showed no detectable in vitro DNA packaging activity. The purified D255E, D255N, D255T, D255V, and D255E/E256D mutant proteins exhibited defective ATP binding and very low or no gp16-stimulated ATPase activity. The nuclease activity of gp17 is, however, retained, albeit at a greatly reduced level. These data define the N-terminal ATPase center in terminases and show for the first time that subtle defects in the ATP-Mg complex formation at this center lead to a profound loss of phage DNA packaging.

Received for publication, July 15, 2005 , and in revised form, October 27, 2005.

^* This work was supported by National Science Foundation grants MCB-110574 and MCB-423528 (to V. B. R.). 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 Present address: Dr. Michael S. Mitchell, HIV DRP Replication Laboratory, NCI-Frederick, Frederick, MD 21702.

² To whom correspondence should be addressed: Dept. of Biology, 103 McCort Ward Hall, The Catholic University of America, 620 Michigan Ave., N.E., Washington, D. C. 20064. Tel.: 202-319-5271; Fax: 202-319-6161; E-mail: rao{at}cua.edu.

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