Defining the bacteriophage T4 DNA packaging machine: evidence for a C-terminal DNA cleavage domain in the large terminase/packaging protein gp17.
Double-stranded DNA packaging in bacteriophage T4 and other viruses occurs by translocation of DNA into an empty prohead by a packaging machine assembled at the portal vertex. Coordinated with this complex process is the cutting of concatemeric DNA to initiate and terminate DNA packaging and encapsidate one genome-length viral DNA. The catalytic site responsible for cutting, and the mechanisms by which cutting is precisely coordinated with DNA translocation remained as interesting open questions. Phage T4, unlike the phages with defined ends (e.g. lambda, T3, T7), packages DNA in a strictly headful manner, and exhibits no strict sequence specificity to initiate or terminate DNA packaging. Previous evidence suggests that the large terminase protein gp17, a key component of the T4 packaging machine, possesses a non-specific DNA cutting activity. A histidine-rich metal-binding motif, H382-X(2)-H385-X(16)-C402-X(8)-H411-X(2)-H414-X(15)-H430-X(5)-H436, in the C-terminal half of gp17 is thought to be involved in the terminase cleavage. Here, exhaustive site-directed mutagenesis revealed that none of the cysteine and histidine residues other than the H436 residue is critical for function. On the other hand, a cluster of conserved residues within this region, D401, E404, G405, and D409, are found to be critical for function. Biochemical analyses showed that the D401 mutants exhibited a novel phenotype, showing a loss of in vivo DNA cutting activity but not the DNA packaging activity. The functional nature of the critical residues and their disposition in the conserved loop region between two predicted beta-strands suggest that these residues are part of a metal-coordinated catalytic site that cleaves the phosphodiester bond of DNA substrate. The data suggest that the T4 terminase consists of at least two functional domains, an N-terminal DNA-translocating ATPase domain and a C-terminal DNA-cutting domain. Although the DNA recognition mechanisms may be distinct, it appears that T4 and other phage terminases employ a common catalytic paradigm for phosphodiester bond cleavage that is used by numerous nucleases.
Department of Biology, The Catholic University of America, 103 McCort Ward Hall, 620 Michigan Ave., N.E. Washington, DC 20064, USA.
Francisco J
FJ
Rentas
Venigalla B
VB
Rao
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D017122:Bacteriophage T4 / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D014764:Viral Proteins / Q000378:metabolism*
D014764:Viral Proteins; C065038:gp17 protein, Enterobacteria phage T4; D001224:Aspartic Acid; D006639:Histidine; D004247:DNA; D004706:Endodeoxyribonucleases; C030894:terminase; D000251:Adenosine Triphosphatases; D003545:Cysteine