PMID:17870092
| Citation |
Ortega, ME, Gaussier, H and Catalano, CE (2007) The DNA maturation domain of gpA, the DNA packaging motor protein of bacteriophage lambda, contains an ATPase site associated with endonuclease activity. J. Mol. Biol. 373:851-65 |
|---|---|
| Abstract |
Terminase enzymes are common to double-stranded DNA (dsDNA) viruses and are responsible for packaging viral DNA into the confines of an empty capsid shell. In bacteriophage lambda the catalytic terminase subunit is gpA, which is responsible for maturation of the genome end prior to packaging and subsequent translocation of the matured DNA into the capsid. DNA packaging requires an ATPase catalytic site situated in the N terminus of the protein. A second ATPase catalytic site associated with the DNA maturation activities of the protein has been proposed; however, direct demonstration of this putative second site is lacking. Here we describe biochemical studies that define protease-resistant peptides of gpA and expression of these putative domains in Escherichia coli. Biochemical characterization of gpA-DeltaN179, a construct in which the N-terminal 179 residues of gpA have been deleted, indicates that this protein encompasses the DNA maturation domain of gpA. The construct is folded, soluble and possesses an ATP-dependent nuclease activity. Moreover, the construct binds and hydrolyzes ATP despite the fact that the DNA packaging ATPase site in the N terminus of gpA has been deleted. Mutation of lysine 497, which alters the conserved lysine in a predicted Walker A "P-loop" sequence, does not affect ATP binding but severely impairs ATP hydrolysis. Further, this mutation abrogates the ATP-dependent nuclease activity of the protein. These studies provide direct evidence for the elusive nucleotide-binding site in gpA that is directly associated with the DNA maturation activity of the protein. The implications of these results with respect to the two roles of the terminase holoenzyme, DNA maturation and DNA packaging, are discussed. |
| Links |
PubMed PMC2082050 Online version:10.1016/j.jmb.2007.07.067 |
| Keywords |
Adenosine Triphosphatases/chemistry; Adenosine Triphosphatases/genetics; Adenosine Triphosphatases/metabolism; Adenosine Triphosphate/metabolism; Amino Acid Sequence; Bacteriophage lambda/genetics; Bacteriophage lambda/metabolism; Binding Sites; DNA Packaging; DNA, Viral/genetics; DNA, Viral/metabolism; Endodeoxyribonucleases/chemistry; Endodeoxyribonucleases/genetics; Endodeoxyribonucleases/metabolism; Fluorescence; Models, Molecular; Molecular Sequence Data; Mutation; Protein Structure, Tertiary; Virus Assembly |
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