Substrate interactions and promiscuity in a viral DNA packaging motor

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Abstract
Viral packaging motors, multimeric ring-shaped ATPases of the ASCE superfamily, mediate filling of the capsid with the viral genome by translocating unidirectionally along nucleic acid in a nucleotide-dependent manner. Aathavan et al. use single-molecule approaches to determine how the motor protein interacts with the phosphate backbone of the nucleic acid during packing of the Bacillus subtilis bacteriophage ϕ29. They find that phosphate charges facilitate — but are not required for — translocation. In fact, the packaging motor is remarkably promiscuous, and even a non-biological polymer can be packaged. Normally, however, the motor interacts on one strand with a phosphate charge about every 10 base pairs. In several viruses and bacteriophages, DNA transport in processes such as genome packaging is dependent on a subset of the ASCE superfamily of protein enzymes consisting of multimeric ringed pumps. Little is known, however, about how these motors engage their nucleic acid substrates. Here, the genome packaging motor of the Bacillus subtilis bacteriophage is studied, revealing that the full mechanochemical cycle of the motor involves two, rather different, phases. The ASCE (additional strand, conserved E) superfamily of proteins consists of structurally similar ATPases associated with diverse cellular activities involving metabolism and transport of proteins and nucleic acids in all forms of life1. A subset of these enzymes consists of multimeric ringed pumps responsible for DNA transport in processes including genome packaging in adenoviruses, herpesviruses, poxviruses and tailed bacteriophages2. Although their mechanism of mechanochemical conversion is beginning to be understood3, little is known about how these motors engage their nucleic acid substrates. Questions remain as to whether the motors contact a single DNA element, such as a phosphate or a base, or whether contacts are distributed over several parts of the DNA. Furthermore, the role of these contacts in the mechanochemical cycle is unknown. Here we use the genome packaging motor of the Bacillus subtilis bacteriophage ϕ29 (ref. 4) to address these questions. The full mechanochemical cycle of the motor, in which the ATPase is a pentameric-ring5 of gene product 16 (gp16), involves two phases—an ATP-loading dwell followed by a translocation burst of four 2.5-base-pair (bp) steps6 triggered by hydrolysis product release7. By challenging the motor with a variety of modified DNA substrates, we show that during the dwell phase important contacts are made with adjacent phosphates every 10-bp on the 5′–3′ strand in the direction of packaging. As well as providing stable, long-lived contacts, these phosphate interactions also regulate the chemical cycle. In contrast, during the burst phase, we find that DNA translocation is driven against large forces by extensive contacts, some of which are not specific to the chemical moieties of DNA. Such promiscuous, nonspecific contacts may reflect common translocase–substrate interactions for both the nucleic acid and protein translocases of the ASCE superfamily1.