1. Biophysics Graduate Group,
2. Jason L. Choy Laboratory of Single-Molecule Biophysics,
3. QB3 Institute, and,
4. Department of Physics, University of California, Berkeley, California 94720, USA
5. Department of Diagnostic and Biological Sciences and Institute for Molecular Virology,
6. Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455, USA
7. Departments of Molecular and Cell Biology, Chemistry, and Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA
8. These authors contributed equally to this work.
9. Present addresses: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94158, USA (K.A.); Department of Physics and Center for Biophysics and Computational Biology, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801, USA (Y.R.C.).

Correspondence to: Carlos Bustamante^1,2,3,4,7 Correspondence and requests for materials should be addressed to C.B. (Email: carlos@alice.berkeley.edu.).

Abstract

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 29 (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.

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