Substrate interactions and promiscuity in a viral DNA packaging motor

Abstract

The ASCE 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 are multimeric ringed pumps responsible for DNA transport in processes including genome packaging in adenoviruses, herpesviruses, poxviruses, and tailed bacteriophages2. While their mechanism of mechanochemical conversion is beginning to be understood3, little is known about how these motors engage their nucleic acid substrates. Do motors contact a single DNA element, such as a phosphate or a base, or are contacts distributed over multiple parts of the DNA? In addition, what role do these contacts play in the mechanochemical cycle? Here we use the genome packaging motor of the Bacillus subtilis bacteriophage φ294 to address these questions. The full mechanochemical cycle of the motor, whose ATPase is a pentameric-ring5 of gene product 16, involves two phases-- an ATP loading dwell followed by a translocation burst of four 2.5-bp steps6 triggered by hydrolysis product release7. By challenging the motor with a variety of modified DNA substrates, we find 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. In addition to 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, non-specific contacts may reflect common translocase-substrate interactions for both the nucleic acid and protein translocases of the ASCE superfamily1.

Single-Stranded DNA Uptake during Gonococcal Transformation

“…Such a cyclic molecular motor would require a well-defined secondary DNA structure for specific binding. For example, the translocation motor required for packaging of DNA into bacteriophage 29 requires electrostatic contacts with adjacent pairs of backbone phosphates spaced every 10 bp on one DNA strand (38). Our results strongly suggest that such a scenario requiring tight coupling is not the underlying mechanism.…”

Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation

“…Removal of the IDs during translocation is at first sight surprising, as the T4 motor can efficiently package DNA labeled with dye covalently attached to the DNA ends or to the bases (19). The Φ29 packaging ATPase is also packaging tolerant of heavily modified duplex DNA (20). And although ID removal is expected and in fact shown for a helicase that separates the DNA strands, this is not expected for a packaging translocase that moves intact B form DNA from outside to inside the prohead.…”

Large Terminase Conformational Change Induced by Connector Binding in Bacteriophage T7

“…Also, the relative motion between terminase subunits has been implicated in the DNA packaging of phage ⌽29 (10). In this case optical tweezers experiments have shown that the interaction between the terminase and the DNA changes during the mechano-chemical translocation cycle (8). Two defined states, the dwell and the burst, characterized by differential contacts between the terminase and the DNA, are involved in the sequential nature of DNA movement (10).…”