Substrate interactions and promiscuity in a viral DNA packaging motor

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 life. 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 bacteriophages. Although their mechanism of mechanochemical conversion is beginning to be understood, 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 varphi29 (ref. 4) to address these questions. The full mechanochemical cycle of the motor, in which the ATPase is a pentameric-ring of gene product 16 (gp16), involves two phases-an ATP-loading dwell followed by a translocation burst of four 2.5-base-pair (bp) steps triggered by hydrolysis product release. 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 superfamily.

Figure 1. Packaging of neutral DNA analogs

(a) A prohead-gp16-ATPase-motor complex bound to a microsphere is held in an optical trap while a micropipette holds a second microsphere bound to DNA containing a modified insert. A tether is formed and packaging is initiated when the beads are briefly brought into close proximity in the presence of ATP. (b) Representative packaging traces of DNA containing 10 bp ds-MeP at a constant load of 5pN. Blue traces show traversal following a pause, and the red trace shows a terminal dissociation event following a pause. Inset are a schematic of the insert, with MeP nucleotides in red and unmodified nucleotides in blue, and the chemical structure of a MeP nucleotide. (c) Force and ATP dependence of traversal probability and pause duration of 10 bp ds-MeP inserts. (d) Traversal probability of 30 bp ds-MeP and DNA-MeP hybrid inserts at 5 pN. (e) Traversal probability of ds-MeP inserts at 5 pN force as a function of insert length. P-values (Two-tailed Fisher exact test) between 9 and 10 bp, and 10 and 11 bp are indicated. (f) Translocation cycle length and footprint size limits from MeP length dependence. This scheme shows the position of a subunit that contacts the DNA before and after a full mechanochemical cycle, i.e. 10 bp. Contact with a single phosphate would produce a drop in traversal probability between 9 and 10 bp ds-MeP whereas contact with two phosphates would produce the observed drop between 10 and 11 bp ds-MeP. In c, d, and e the traversal probability is plotted using the Laplace best estimator, with 95% confidence intervals from the adjusted Wald method, and error bars of pause durations are the SEM.

Figure 2. High resolution dynamics at neutral-DNA insert

(a) Base-pair-scale dynamics at 10 bp of ds-MeP consist of two classes of sub-pauses, upstream and downstream pauses, separated by attempts and punctuated by slips and repackaging events. (b) A cartoon model of the dynamics of these pauses with average lifetimes and inter-conversion probabilities. The full statistics of these states are in Supplementary Table 3. (c,d) Histograms of upstream and downstream pause durations. The distributions have n_min values—the ratio of the mean squared to the variance—of 1.1 ± 0.1 (s.d.) and 1.3 ± 0.4 (s.d.), respectively; and are thus well-described by single exponential decays. (e) Histogram of position changes in the upstream pause. (f) Histogram of distance between upstream and downstream pauses.

Figure 3. Substrate promiscuity

(a–d) The force dependence of traversal probabilities and pause durations of different modified DNA substrates. (a) 10 nt ds-abasic phosphate backbone. (b) 20 nt ssDNA (poly-AC) on 5’-3’ and 3’-5’ strands. (c) 10bp bulge (poly-AC) on 5’-3’ and 3’-5’ strands. (d) ds-linker. The traversal probabilities are plotted using the Laplace best estimator, with 95% confidence intervals from the adjusted Wald method.The error bars of the pause durations are the SEM. (The results for other sizes of bulges and gaps are listed in Supplementary Table 2.)

Figure 4. The Motor-DNA Contacts

The relative importance of the motor-DNA contacts are marked using pymol with a quantitative color scale with magnitudes inferred from the traversal probabilities at 5 pN for the measured modifications (Figure 1–Figure 3; Supplementary Table 2, Supplementary Discussion; Supplementary Figure 2). The units of “contact importance” correspond to the inverse of the distance over which the removal of the specific moiety would reduce the traversal probability to 50% (Supplementary Discussion.) The mechanochemical phase of the motor as it moves along the DNA is indicated to the right.