© 2006 Nature Publishing Group
Mechanism of DNA translocation in a
replicative hexameric helicase
Eric J. Enemark1& Leemor Joshua-Tor1
The E1 protein of papillomavirus is a hexameric ring helicase belonging to the AAAþ family. The mechanism that
couples the ATP cycle to DNA translocation has been unclear. Here we present the crystal structure of the E1 hexamer
with single-stranded DNA discretely bound within the hexamer channel and nucleotides at the subunit interfaces. This
structure demonstrates that only one strand of DNA passes through the hexamer channel and that the DNA-binding
hairpins of each subunit form a spiral ‘staircase’ that sequentially tracks the oligonucleotide backbone. Consecutively
grouped ATP, ADP and apo configurations correlate with the height of the hairpin, suggesting a straightforward DNA
translocation mechanism. Each subunit sequentially progresses through ATP, ADP and apo states while the
associated DNA-binding hairpin travels from the top staircase position to the bottom, escorting one nucleotide of
single-stranded DNA through the channel. These events permute sequentially around the ring from one subunit to the
Replication initiation only occurs at specific DNA sites (origin of
replication, ori), and can only occur once per cell division. Initiation
begins with binding of ori by an origin-recognition protein such as
bacterial DnaA (reviewed in ref. 1) or yeast ORC (reviewed in ref. 2).
Subsequently, a helicase is assembled at ori and DNA polymerase(s),
primase(s) and other factors are recruited to form the replisome,
which processively synthesizes DNAcomplementarily to the template.
In eukaryotes replication initiation involves many proteins. Even in
Escherichia coli, at least three proteins (DnaA, DnaB and DnaC)1are
required just to establish the replicative helicase at ori. Small DNA
viruses such as papillomavirus, SV40 and AAV (adeno-associated
virus) use a single initiator protein for origin recognition, helicase
loading and helicase activity itself.
The viral initiator proteins E1, large T-antigen and Rep (from
papillomavirus, SV40 and AAV, respectively) belong to helicase
superfamily III (SF3; reviewed in ref. 3) and are members of the
AAAþ (ATPases associated with cellular activities) family4. These
helicases form hexameric rings that are believed to encircle substrate
DNA and unwind it with 3
resemble those of bacteriophage T7 gene product 4 (T7gp4) and
E. coli DnaB, two hexameric replicative helicases that exhibit the
to encircle only one of the two DNA strands10,11. The strand
displacement assays used to determine the polarity of SF3 helicases
indicates that the hexamers will load upon a single-stranded 3
tail7,12, suggesting that the ring encircles this strand while sterically
excluding the other. A mechanism where double-stranded DNA
(dsDNA) passes through the channel after assembly at the ori was
proposed for these helicases13,14. However, the crystal structures of
SV40 T-antigen (Tag)13,14demonstrate channel diameters of ,7A˚in
the ATP-bound state and ,15A˚in the apo state in the AAAþ
dsDNA, suggesting that only ssDNA can be accommodated.
Papillomavirus and SV40 have a closed circular genome, and
consequently no ends are availablefor loading an intact ring. Whereas
most DNA helicases require a region of ssDNA for entry, E1 and Tag
can initiate unwinding from completely dsDNA, apparently by
0polarity5–7. These properties
0polarity8,9. Both T7gp4 and DnaB have been shown
causing helix melting and entry of the DNA helicase onto a single-
stranded region. Sequence similarity between the carboxy-terminal
halves of E1 and SV40 T-antigen, which contain the AAAþ domain,
is significant. The amino-terminal halves of the proteins, which
contain the DNA-binding domain (DBD), share little sequence
similarity, but their structures are highly similar15,16, reinforcing the
likelihood that they use analogous mechanisms. The closed ring
topology of their genomes suggests that the helicase is loaded as an
open ring17, as proposed for DNA processivity clamps18, or as
monomers sequentially until a hexameric ring encircling the DNA
binding (origin binding activity)to aspecific single-stranded binding
and unwinding (helicase activity) via a transition of stoichiometry
from a dimer to a double hexamer through a series of discrete
intermediate oligomeric states assembled directlyupon progressively
distorted6,19–21and ultimately melted origin DNA22,23. To determine
the topological and atomic details of DNA coordination by this
group of ring helicases, and to elucidate features of the DNA
translocation mechanism, we have determined the crystal structure
of a bovine papillomavirus (BPV) E1 fragment comprising the
AAAþ and oligomerization domains in complex with ssDNA,
ADP and Mg2þ.
The crystal consists of two hexamers (subunits A–F for hexamer 1
and G–L for hexamer 2), each encircling a single strand of DNA
(Fig. 1). The six oligomerization domains form a rigid collar with
near proper six-fold rotational symmetry. The arrangement of the
AAAþdomainsresemblesthearrangement intheTag hexamer14and
in the predicted human papillomavirus type 18 (HPV-18) E1 AAAþ
this case they deviate significantly from proper six-fold symmetry.
Most of the protein–DNA interactions occur at hairpins located at
the interior of the channel, which narrows to ,13A˚in diameter.
These hairpins resemble a spiral staircase as they sequentially track
the sugar-phosphate backbone of the ssDNA in a right-handed
helical arrangement of a consistent set of interactions (Fig. 2). The
1W. M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
Vol 442|20 July 2006|doi:10.1038/nature04943
© 2006 Nature Publishing Group
subunits have varying modes of Mg2þ–ADP coordination, and each
P-loop appears to have an associated ADP molecule except for
All of the residues observed to interact with ssDNA are located at the
interior of the hexameric ring on the AAAþ domains (Fig. 2). The
while the H507 main-chain amide proton forms a hydrogen bond
with the ssDNA phosphate of an adjacent nucleotide. The aliphatic
der Waals interactions with the ssDNA sugar moiety linking these
two phosphates. This set of interactions permutes sequentially
around the hexameric ring with concurrent unitary ssDNA nucleo-
tide progression between the subunits. The 5
directed towards the N-terminal oligomerization domains, whereas
interactions are consistent between the two hexamers with one
notable exception. One hexamer has five of the subunit hairpins
engages all six subunit hairpins. The details of this difference
illustrate alogical transitionwithin a cyclic translocation mechanism
0end of the ssDNA is
0end is directed towards the C terminus. The protein–DNA
Intersubunit interactions and ADP binding
The most robust intersubunit interactions occur between the oligo-
merization domains through consistent interactions at each interface.
The interactions between the AAAþ domains either mediate nucleo-
tide binding or are involved in ‘staircasing’ the hairpins. As with Tag,
the RFC (replication factor C) clamp loader, and other multisubunit
AAAþ protein complexes, the amino acids responsible for nucleotide
coordination and hydrolysis are located on adjacent subunits. One
contains the Walker A, Walker B and sensor-1 motifs. The adjacent
subunit provides additional elements such as the arginine finger. For
the E1–DNA hexamer, the intersubunit arrangement and separation
varies. Consequently, the atomic details of nucleotide binding vary
between the subunits (Fig. 3). In all cases, the Walker A and B motifs
are consistently structured and generally coordinate the diphosphate
group of an ADP molecule and an associated Mg2þion. In contrast,
the adjacent subunit has threefundamental forms that correlate with
the DNA-binding hairpin in the staircase.
Form Iis present at three of the interfaces in hexamer 1 and at two
of the interfaces in hexamer 2 (see also Supplementary Fig. S1 and
Supplementary Table S1). This form involves an intricate network of
interactions between adjacent subunits (Fig. 3). Based on three
factors, this form of nucleotide coordination is classified as ATP-
type: (1) simultaneous engagement of all the AAAþ components
implicated in ATP coordination and hydrolysis; (2) all of these
elements structurally align with equivalents in the (Tag-ATP)6
structure (Supplementary Fig. S2); and (3) a chloride ion sits at
the anticipated position for an ATP g-phosphate in three of the five
cases (difference density is also observed at 2.5j and 3j in the other
two cases but has not been modelled). The positions of K425 and
R538 (the arginine finger) are major factors that define this nucleo-
tide coordination mode. The ammonium group of K425 concur-
rently binds the a- and b-phosphate in a position very similar to the
sensor-2 arginine, R217, of RuvB, an AAAþ protein involved in
Holliday branch migration. As a result, we classify this lysine as
‘sensor-2’. The arginine finger, R538, simultaneously binds a Walker
B aspartate of the first subunit, D479, and a water molecule that
coordinates the Mg2þion. Notably, the guanidinium group of R538
Figure 1 | Structure of the E1
hexameric helicase in complex with
ssDNA and ADP. a, b, Ribbon
representations of the two E1
helicase hexamers viewed
the channels. Individual subunits
are colour-coded with hexamer1 on
crystal lattice interaction between
the hexamers is illustrated (a). The
oligomerization domains form a
rigid collar, located between the
AAAþ domains in a and projected
the central channel and the ADP
molecules at subunit interfaces are
depicted in stick representation.
Images were prepared with
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© 2006 Nature Publishing Group
is appropriately positioned to interact with the g-phosphate of an
ion also interacts with sensor-1 of the first subunit, N523. Several
residues on a connected pair of helices participate in this coordi-
nation. Arginine R493, denoted here as ‘sensor-3’, interacts with
D489 as well as the Walker B aspartate, D479, and sensor-1, N523.
Tyrosine Y499 interacts with the ADP a-PO4.
Form II uses the Walker A and B motifs of one subunit, but only
Y499 of the adjacent subunit (Fig. 3). In this form, most of the ATP-
type interactions are absent and display flexibility. R493 interacts
with Walker B D479 and sensor 1 N523 of the neighbouring subunit
for the interface between subunits D and E of hexamer 1 and the I–J
interface of hexamer 2. However, it is too distant for the E–F
(hexamer 1) and J–K(hexamer 2) interfaces(see also Supplementary
Fig. S1). A striking difference between this form and the ATP type
described above is the sandwiching of Y534 between R493 and R538
(arginine finger). This interaction places R538 too distant to interact
with a g-PO4of an ATP molecule. The greatly reduced interactions
and complete exclusion of the arginine finger led us to classify this as
‘ADP-type’ coordination. No Cl2ions are observed in this form.
FormIIIdoesnotuseany residues oftheadjacentsubunitandalso
form has been classified as ‘apo’ due to the total lack of involvement
of the adjacent subunit. In the apo-type coordination, an ADP
molecule is usually present at the Walker A and B motif regions,
but it is coordinated differently than above. In the K–L interface, the
a-PO4and b-PO4of the ADP molecule adopt a reverse orientation
relative to the other subunit interfaces.
Correlation of nucleotide state with hairpin height
The DNA-binding hairpins form a right-handed staircase that
sequentially tracks the ssDNA backbone. The vertical position on
the staircase for the hairpin of a given subunit correlates with the
type of nucleotide coordination. The subunits that participate in
ATP-type coordinations place their hairpins at the top of the stair-
case; the hairpins of apo-type subunits occupy the bottom of the
staircase. The hairpins of ADP-type subunits lie at the intermediate
positions. The staircase is maintained by a consistent set of inter-
actions (Fig. 2) between the hairpins of adjacent subunits involving
K506, which also contacts a ssDNA phosphate as described above.
The ammonium group of this lysine interacts with three sites on the
adjacent hairpin: the acidic group of D504 and the main-chain
carbonyl groups of R505 and K508. These staircasing elements are
these proteins to exhibit the same sets of interactions and form a
staircase of hairpins when ssDNA and multiple nucleotide states are
present simultaneously (Supplementary Fig. S3). Previous crystal
state13,14, did not display a staircase of hairpins. Interestingly, a
hexameric ring structure of T7gp4, which had multiple nucleotide
states but lacked DNA25, also demonstrated a staircase of the DNA-
binding hairpins via a lysine (K467, chain C) and an acidic residue
with assigned ATP-type coordination. Also, these staircasing elements
with T7gp4 (Supplementary Fig. S4). The adoption of a staircase
by DNA-binding hairpins through a set of interactions involving a
Figure 2 | DNA binding in the E1 hexameric
helicase. a–c, Interactions involving the
AAAþ hairpins and ssDNA shown as a
perpendicular views (b, c) for hexamer 1.
K506 coordinates a ssDNA phosphate, and
the main-chain amide of H507 interacts with
the phosphate of an adjacent nucleotide.
K506 also mediates interactions with three
sites on the adjacent hairpin to form the
staircase of hairpins—the side chain of D504
and the main chain carbonyl groups of R505
and K508 (the latter two side chains are
omitted for clarity). Hydrogen bonds are
drawn as dotted (a) or dashed (b–d) lines.
Hairpins of the individual subunits are
colour-coded as in Fig. 1. In b and c only
H507 and K506 side chains are shown. The
letters in parentheses identify the subunits.
Other components of the protein are
in light blue. d, Same asc with superimposed
Fo2 Fcdifference electron density
model. The electron density is contoured at
3j (red) and at 6j (blue).
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© 2006 Nature Publishing Group
basic and an acidic residue of a neighbouring subunit may be a
fundamental feature of hexameric helicases.
The correlation of nucleotide state with vertical position of the
DNA-binding hairpins is consistent with previous structural obser-
DNA, the major difference here is that these states are all present in
the same hexamer.
The crystal structure of hexameric T7gp4 bound to the non-
hydrolysable ATP analogue ADPNP in the absence of DNA also
exhibits multiple nucleotide-binding configurations with an associ-
related set of three sites is observed, two with associated ADPNP
molecules (ATP-type and ADP-type) and one empty.
A coordinated escort mechanism for DNA translocation
The hexameric structures determined here display varying nucleo-
tide coordination modes that correlate with the vertical position of
the associated DNA-binding hairpins. The ssDNA nucleotides are
Figure 3 | Intersubunit interactions and nucleotide binding. a, b, The
hydrogen-bonding networks in the intersubunit nucleotide coordination
types shown as a schematic (a) and in stereoview (b). The subunit
containing the Walker A, Walker B and sensor-1 motifs is coloured yellow;
the adjacent subunit containing the arginine finger is coloured cyan. Mg2þ
ions are coloured purple and Cl2ions are green. The ATP-like coordination
(top) has multiple interactions and engages R538 and K425. A Cl2ion is
generally observed at the anticipated position for a g-PO4. The ADP-like
and R538 precludes the interactions observed in the ATP-like case. K425 is
too distant to interact with the nucleotide. Successive ADP-type
configurations (middle) show increasing separation between the subunits.
The apo configuration is shown at the bottom.
NATURE|Vol 442|20 July 2006
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arranged with a 1 nucleotide per subunit increment. As a result, a
straightforward and compelling DNA translocation mechanism
integrating these details can be inferred (Fig. 4). Each DNA-binding
hairpin maintains continuous contact with one unique nucleotide of
ssDNA and migrates downward via ATP hydrolysis and subsequent
ADPrelease at the subunit interfaces. ATP hydrolysis occurs between
subunits located towards the top of the staircase, while ADP release
movements of the hairpins are coordinated by the staircasing
interactions described above. The hairpin at the bottom of the
staircase releases its associated ssDNA phosphate to conclude its
journey through the hexameric channel. Upon binding a new ATP
next available ssDNA phosphate, initiating its escorted journey
through the channel and repeating the process. For one full cycle
of the hexamer, each subunit hydrolyses 1ATP molecule, releases 1
ADP molecule and translocates 1 nucleotide through the interior
channel. A full cycle, therefore, translocates 6nucleotides with
associated hydrolysis of 6ATPs and release of 6ADPs.
Both hexamers display a noticeable gap between the AAAþ
domains located at the top and bottom positions of the staircase
(Fig. 1b). This interface is where the most sizeable movements occur
in the mechanism proposed here. Upon ATP binding, the subunit at
the bottom of the staircase moves to the top position and closes this
gap. Simultaneously, a new gap opens between this subunit and the
previous subunit (now bottom). The two hexamers in the crystal
display different configurations at this interface, as demonstrated by
hairpins engaged in ssDNA coordination (Supplementary Table S1).
For hexamer 1, an ATP-type configuration is observed between
subunits A and B, but the hairpin of subunit A is not engaged in
ssDNA coordination. Subunit A is consistent with a subunit that has
recently bound an ATP molecule, but has not yet bound DNA (see
also Fig. 2b, c). This state is not present in hexamer 2, where the
subunit at the top of the staircase has an ATP state and binds to
ssDNA. Hexamer 2, however, displays two empty nucleotide states
whereas hexamer 1 has only one. Subunit L is the lowest on the
hexamer 2 staircase and has no counterpart in hexamer 1. It
represents the state that directly precedes ATP binding. This is the
than the other subunits.
This mechanism has some similarities to the previously proposed
operation of T7gp4. For the previous T7gp4 proposal, successive
loops also bind to successive nucleotides but pass them through the
channel by handing them off from one loop to the next in a “bucket
nearly fixed height as the individual nucleotides are passed down-
wardfrom one loop to the next. In contrast, in the escort mechanism
proposed here, each hairpin maintains a continuous set of inter-
actions with one nucleotide, and the entire unit collectively
migrates downward. We suggest that the T7gp4 hairpins also co-
migrate with a given nucleotide in an analogous coordinated escort
mechanism. The assigned nucleotide configurations in the crystal
structure of T7gp4 in complex with ADPNP25place the DNA-
binding loops of the ATP-assigned coordination farthest from the
primase domains, while the empty state places them closest, in
agreement with the probable translocation of DNA towards the
affinity for DNA than the ADPand empty states27. The same relative
order of affinities in the E1 hexamer would lead to an additional
driving force for the coordinated escort mechanism of cyclic DNA
translocation because subunits at the top of the staircase would have
a higher affinity for DNA (more prone to bind) than those at the
bottom of the staircase (more prone to release). Binding of a new
ATP molecule by a subunit in the empty state at the bottom of the
staircase would serve not only to return it to the top of the staircase,
but also to increase its affinity for DNA.
The mechanism described here directly couples ATP hydrolysis to
DNA translocation by using ATP hydrolysis to drive the sequential
movement of the hairpins and pump ssDNA in that direction. This
mechanism is consistent with the demonstrated 3
SF3 helicases and the observed polarity of the DNA in the structure
with the 5
was proposed for Tag13, where the same correlation of hairpin height
with nucleotide configuration was identified in the absence of DNA.
DNA was proposed to translocate from the empty associated state
(bottom) towards the ATP-associated state (top). Adrawback of that
mechanism is that it does not derive any work from ATP hydrolysis.
Such a mechanism is energetically driven by ATP binding. ATP
hydrolysis would only serve to facilitate return to the empty state.
0end located towards the top (oligomerization domain
Oligomerization domain as processivity factor
The rigid collar formed by the oligomerization domains could serve
as a single-stranded equivalent of double-stranded processivity
factors such as polymerase sliding clamps like PCNA. Whereas
PCNA holds dsDNA topologically together, the static ring formed
by six oligomerization domains in the E1 helicase keeps two strands
topologically apart. This functionwould be important to prevent the
ring from falling off the substrate DNA because the AAAþ domains
have relatively weak interactions between the subunits, including a
significant gap between the subunits with the bottom and top
hairpins. The robust intersubunit interactions of the ring formed
by the oligomerization domains ensure that the hexamer continues
to surround the given single strand.
A translocation mechanism in which ATP is hydrolysed sequentially
from one subunit to the next with the staircase migrating downward
may constitute a general mechanism for hexameric helicases.
Although the three nucleotide-binding configurations observed in
the structure define relative positions for the ATP, ADP and empty
states in a coordinated escort mechanism, the precise details regard-
ing the sequence and timing of ATP hydrolysis, phosphate release,
ADP release and ATP binding in this mechanism remain to be
determined. This mechanism might not operate by one exclusive
timing of these events; slight variations might operate in parallel
as described for T7gp4 (ref. 28). The localization of the double-
stranded/single-stranded fork junction in this complex also remains
Figure 4 | Cartoon depiction of a coordinated escort mechanism for the E1
hexameric helicase. Each subunit is depicted as a wagon (or boxcar),
colour-coded as in Fig. 1. Each wagon transports one DNA nucleotide from
the right side to the left. Staircasing interactions are depicted by the wagon
couplers. ATP hydrolysis and ADP release occur along the path as depicted
by the nucleotide coordination type between the wagons. The red, leftmost
wagon ejects its associated DNA nucleotide and returns to the right side
upon binding a new ATP molecule. This wagon then picks up the next DNA
nucleotide of the series, couples to the purple wagon, and carries its cargo
towards the left. Figure prepared by J. Duffy.
NATURE|Vol 442|20 July 2006
© 2006 Nature Publishing Group
unclear. This species may involve the DBD, which in this model
is anticipated to be proximal to the dsDNA entering the complex.
The assembly of the helicase at the ori relies upon the DBD. The
ultimate helicase species established at the ori is a double hexamer29.
Our structure suggests that the double hexamer consists of two
individual hexamers that encircle opposing single strands. The heli-
case activity of this species results from unidirectional translocation
along the encircled strand with steric exclusion of the other, as
described for other helicases such as the RNA helicase NPH-II30.
The mechanism described here can be used to establish two different
species where the hexamers remain associated or move apart, as seen
by electron microscopy31,32(see Supplementary Fig. S5). Further
studies are necessary to address these issues.
Complex preparation. A fragment encoding BPV-1 E1 residues 306–577 was
plasmid template provided by A. Stenlund, and cloned into pGEX-4T-1
(Amersham). The GST fusion protein was expressed in E. coli and purified in
a manner analogous to methods described previously for other E1 GST fusion
proteins15, and was concentrated in the presence of 5mM ADP and 200mM
oligonucleotide (13-mer), and immediately used for crystallization trials.
Crystals grew at 178C by the hanging-drop method with a well solution
favourable sample was transferred to liquid nitrogen and moved to beamline
X29 for data collection. Data collection statistics are shown in Supplementary
Structure determination and refinement. The structure was solved by mol-
ecularreplacementwith theprogramPHASER33by placing12 copiesofa hybrid
search model consisting of the previously reported helicase domain of HPV-18
E1 (ref. 24) (Protein Data Bank code 1TUE) and the helicase domain of BPV E1
(E.J.E., T. J. Takara, A. Stenlund and L.J., unpublished structure of E1(368–577)
and refinement can be found in Supplementary Information.
Received 8 April; accepted 1 June 2006.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements We thank H. Robinson (beamline X29) and A. He ´roux
(beamline X26C) for support with data collection at the National Synchrotron
Light Source (NSLS) at Brookhaven National Laboratory. We also thank
G. Hannon and A. Gann for critical reading of the manuscript, and B. Stillman,
N. Tolia and members of the Joshua-Tor laboratory for discussions. The NSLS is
supported by the US Department of Energy, Division of Materials Sciences and
Division of Chemical Sciences. This work was supported by an NIH grant to L.J.
Author Information Coordinates and structure factors are deposited in the
Protein Data Bank under accession code 2GXA. Reprints and permissions
information is available at npg.nature.com/reprintsandpermissions. The authors
declare no competing financial interests. Correspondence and requests for
materials should be addressed to L.J. (email@example.com).
NATURE|Vol 442|20 July 2006