Molecular Cell, Vol. 10, 647–657, September, 2002, Copyright 2002 by Cell Press
DnaB Drives DNA Branch Migration and Dislodges
Proteins While Encircling Two DNA Strands
Daniel L. Kaplan1,3 and Mike O’Donnell1,2
Laboratory of DNA Replication
2
Howard Hughes Medical Institute
Rockefeller University
New York, New York 10021
1
Summary
DnaB is a ring-shaped, hexameric helicase that unwinds the E. coli DNA replication fork while encircling
one DNA strand. This report demonstrates that DnaB
can also encircle both DNA strands and then actively
translocate along the duplex. With two strands positioned inside its central channel, DnaB translocates
with sufficient force to displace proteins tightly bound
to DNA with no resultant DNA unwinding. Thus, DnaB
may clear proteins from chromosomal DNA. Furthermore, while encircling two DNA strands, DnaB can
drive branch migration of a synthetic Holliday junction
with heterologous duplex arms, suggesting that DnaB
may be directly involved in DNA recombination in vivo.
DnaB binds to just one DNA strand during branch migration. T7 phage gp4 protein also drives DNA branch
migration, suggesting this activity generalizes to other
ring-shaped helicases.
Introduction
The replicative helicase of E. coli, DnaB, is a ring-shaped
hexamer that encircles the lagging strand while unwinding DNA at a replication fork (LeBowitz and McMacken,
1986). Functional homologs in other systems include
the T7 phage gp4 and the T4 phage gp41 proteins.
Electron microscopy studies show that these hexameric
replication fork helicases are ring-shaped with a central
channel of ⵑ25–40 Å in diameter (Dong et al., 1995;
Egelman et al., 1995; Martin et al., 1998).
It has been shown by a variety of techniques that
single-stranded DNA passes through and binds to the
central channel of these ring-shaped helicases (Egelman et al., 1995; Hacker and Johnson, 1997; Kaplan,
2000; Morris and Raney, 1999). There are two crystal
structures of related proteins that are ring-shaped,
wherein the putative DNA binding site is inside the central channel (Singleton et al., 2000; Niedenzu et al., 2001).
These helicases can unwind duplex DNA in vitro if the
DNA resembles a replication fork. In other words, the
duplex DNA must have both 5⬘ and 3⬘ single-stranded
extensions, or “tails,” at one end of the duplex (Ahnert
and Patel, 1997; Kaplan and Steitz, 1999; LeBowitz and
McMacken, 1986; Richardson and Nossal, 1989). Furthermore, DnaB and its homologs translocate along single-stranded DNA and unwind duplex DNA with a distinct polarity in the 5⬘ to 3⬘ direction (Kaplan, 2000;
LeBowitz and McMacken, 1986; Richardson and Nossal,
1989). Recent studies of DnaB from Thermus aquaticus
3
Correspondence: [email protected]
demonstrate that the protein first binds to the 5⬘ singlestranded tail region with the DNA positioned inside the
central channel. With the 3⬘ tail positioned outside the
channel, the helicase then translocates with 5⬘ to 3⬘
polarity and unwinds the double-stranded DNA. These
conclusions are supported by studies of T4 gp41 and T7
gp4, leading to a model for unwinding by these helicases
(Figure 1A) (Ahnert and Patel, 1997; Hacker and Johnson, 1997; Kaplan, 2000).
When T. aquaticus DnaB tracks along ssDNA and then
encounters a duplex with no 3⬘ tail, the DnaB moves
off the ssDNA in order to encircle both strands and
translocate along the dsDNA (Figure 1B) (Kaplan, 2000).
Thus, when the 3⬘ tail is long or bulky, the strand passes
outside of DnaB’s ring, and unwinding occurs. When
the 3⬘ tail is absent, DnaB slides up over both strands
such that they pass through the central channel of the
protein. Because unwinding does not occur when two
strands pass through the central channel of DnaB, it
was unclear if this movement was by passive diffusion
or active translocation.
In this study, we find that translocation of DnaB along
DNA with two strands positioned in the central channel
is an active process, as it generates sufficient force to
displace a tightly bound protein from DNA. Surprisingly,
we find that DnaB couples this force to drive branch
migration of a synthetic Holliday junction, and we have
determined the mechanism of this activity. During
branch migration, DnaB encircles two DNA strands, but
binds to only one of these strands. Active translocation
in the 5⬘ to 3⬘ direction along this strand causes branch
migration.
Results
E. coli DnaB Does Not Unwind Duplex DNA
Bearing Only a 5ⴕ Tail
It was previously demonstrated that T. aquaticus DnaB
can unwind duplex DNA that resembles a replication
fork, having both 5⬘ and 3⬘ tails on one end of the duplex
(forked duplex, Figure 1A) (Kaplan and Steitz, 1999).
However, T. aquaticus DnaB cannot unwind duplex DNA
that bears only a 5⬘ single-stranded DNA extension, or
tail (Figure 1B) (Kaplan and Steitz, 1999). To determine
if E. coli DnaB functions in a similar manner, duplex DNA
with a fork (Figure 1A) or with a 5⬘ tail (Figure 1B) was
constructed using synthetic DNA oligonucleotides.
These substrates contain a duplex region of 50 base
pairs and single-stranded tails of 30 dT. E. coli DnaB
was incubated with 32P-labeled DNA substrate in the
presence of ATP, and the reaction was analyzed by
native polyacrylamide gel electrophoresis.
E. coli DnaB can unwind a forked duplex (Figures 1A
and 1C), but it cannot unwind a duplex that has only a
5⬘ tail (Figures 1B and 1C). These results are similar to
those observed using T. aquaticus DnaB (Kaplan and
Steitz, 1999).
Study of the T. aquaticus DnaB on a substrate lacking
a 3⬘ tail (i.e., Figure 1B) demonstrated that the enzyme,
Molecular Cell
648
passive diffusion along DNA, the movement is not coupled to ATP hydrolysis and it cannot exert force. In
active translocation, the movement is coupled to ATP
hydrolysis and it exerts force during movement. Next,
an experiment is designed to determine if E. coli DnaB
translocates on duplex DNA and to distinguish between
passive and active processes.
Figure 1. DnaB Displaces Protein from DNA While Encircling One
Strand or Two Strands
(A and B) Synthetic duplex substrates contain a 5⬘ ssDNA tail that
serves as an assembly site for the ring-shaped DnaB hexamer. DNAs
are labeled with 32P (asterisk). DnaB was incubated with substrate
containing: (A) fork (5⬘ and 3⬘ ssDNA tails) or (B) only a 5⬘ ssDNA
tail. Positions of substrate and product are indicated to the right of
each native gel.
(C) Quantitation of results of (A) (squares) and (B) (circles).
(D) DnaB displaces 32P-EBNA1PK from 5⬘-tailed duplex DNA. The
scheme illustrates that 32P-EBNA1PK displaced from the synthetic
substrate by DnaB is trapped by a plasmid which contains 24 EBNA1
sites.
(E) Quantitation of the results in (D) and similar experiments. The
oligonucleotides used to construct these substrates are detailed in
Supplemental Table S1 at http://www.molecule.org/cgi/content/full/
10/3/647/DC1.
upon encountering the duplex, actually transits from
the ssDNA to the duplex such that both strands pass
through the central channel (Kaplan, 2000). As T. aquaticus DnaB moves across duplex DNA, it does not lead
to unwound DNA products. It has not been established
whether the helicase, as it moves along dsDNA, does
so by passive diffusion or by active translocation. In
DnaB Displaces Protein Bound to Duplex DNA
with a 5ⴕ Tail
To address whether DnaB movement along dsDNA is
passive or active, we asked whether E. coli DnaB can
displace a protein that binds tightly to duplex DNA. If
DnaB translocation along duplex DNA is passive, it
should not displace bound protein, but if DnaB movement is an active process, it may be capable of displacing protein from DNA. In the following experiment, we
used EBNA1, the origin binding protein of Epstein Barr
virus, as it is known to bind tightly to its canonical DNA
binding site (Frappier and O’Donnell, 1991). Furthermore, a modified form of EBNA1 that bears a recognition
site for cAMP-dependent protein kinase, called EBNA1PK, can be radioactively labeled while still retaining
its tight DNA binding capacity (Kelman et al., 1995b).
To determine if DnaB can displace protein from
DNA while DnaB encircles two DNA strands, we first
incubated radioactively labeled EBNA1PK protein
(32P-EBNA1PK) with excess duplex DNA bearing a 5⬘ tail
(Figure 1D). We expect that DnaB will migrate along the
ssDNA tail and then move along the duplex by encircling
both DNA strands. If this translocation process is active,
DnaB may displace 32P-EBNA1PK. However, the displaced 32P-EBNA1PK can simply rebind the DNA. Therefore, to detect 32P-EBNA1PK displacement, we added
excess plasmid DNA that contains 24 binding sites for
EBNA1 (pGEMoriP). This plasmid acts as a trap to capture 32P-EBNA1PK as it is released from the 5⬘-tailed duplex DNA. The reaction was analyzed by electrophoresis
in a native agarose gel, which separates substrate
(32P-EBNA1PK bound to the synthetic tailed duplex) from
product (32P-EBNA1PK bound to plasmid). The results
demonstrate that 59% of the 32P-EBNA1PK is displaced
by E. coli DnaB in 16 min (Figure 1D, gel; Figure 1E,
open circles). Furthermore, the DNA is not unwound
during this reaction, either in the presence or absence
of 32P-EBNA1PK (data not shown). This ability to displace
tightly bound protein indicates that when DnaB translocates along duplex DNA without unwinding it, the movement is an active process and is not simple diffusion.
We performed several control experiments to confirm
that EBNA1 displacement is caused by active DnaB
translocation. Very little of the 32P-EBNA1PK is released
from duplex DNA when ATP is not added to the reaction
(Figure 1E, open squares) or when DnaB is not added
to the reaction (Figure 1E, gray diamonds). However,
when the duplex DNA is forked (as in Figure 1A), DnaB
rapidly displaces the 32P-EBNA1PK from the DNA (Figure
1E, closed circles). During this reaction, the DNA is unwound, but unwinding is markedly slowed by the presence of EBNA1PK (data not shown). Protein displacement
by DnaB during unwinding is expected because DnaB,
like all helicases, actively translocates along DNA during
unwinding (LeBowitz and McMacken, 1986). (The rate
DnaB Drives DNA Branch Migration
649
Figure 2. Encounter of DnaB with Holliday
Junctions
DnaB is incubated with ATP and synthetic
Holliday junctions containing either (A) a 5⬘
tail, (B) a fork (5⬘ and 3⬘ tails), (C) a 3⬘ tail, or
(D) no ssDNA tail. DnaB is incubated with a
5⬘-tailed Holliday junction and (E) AMP-PNP
or (F) various nucleotide cofactors (8 min).
The diagrams and arrows to the right and
left of each gel indicate the position of each
product in the gel as determined by analysis
of standards in each gel.
of protein displacement is slower than that of unwinding
in the absence of protein.) When the duplex DNA bears
no tails, DnaB is inefficient in displacing 32P-EBNA1PK
(Figure 1E, closed triangles), consistent with earlier studies showing that DnaB is slow to load onto blunt-ended
dsDNA (Kaplan, 2000).
DnaB Drives Branch Migration of a Synthetic
Holliday Junction
The discovery that DnaB actively translocates along
dsDNA suggested to us that DnaB might be capable of
driving branch migration of a Holliday junction. To test
this idea, we constructed a synthetic Holliday junction
(also called four-way junction or X junction) bearing a
5⬘ tail composed of 30 dT (for DnaB loading) by annealing
four oligonucleotides (Figure 2A). This synthetic Holliday
junction (lacking the 5⬘ tail) has been used to study
proteins that catalyze branch migration of Holliday junctions (Karow et al., 2000). The four duplex DNA arms
are each 25 base pairs, and the 6 base pairs closest to
the center are homologous; thus, spontaneous branch
migration may occur within this central region. However,
the 19 base pairs furthest from the center of the junction
are heterologous, thereby preventing spontaneous
branch migration outside of the central region.
To test DnaB for ability to drive branch migration of
the Holliday junction substrate, we radiolabeled one
strand to follow products in a native gel (strand 1; see
Figure 2A) and then incubated the substrate with DnaB
and ATP. The substrate is rapidly converted to several
product species (Figure 2A). As will be explained further
below, these products are the result of DnaB-catalyzed
branch migration.
We also constructed a Holliday junction with two single-stranded tails at one end to produce a “forked” Holliday junction (Figure 2B). The helicase activity of DnaB
should unwind strand 1 of this substrate. The results in
Figure 2B show a product profile that is explained mainly
by DnaB-catalyzed unwinding (see Supplemental Figure
S1 at http://www.molecule.org/cgi/content/full/10/3/
647/DC1 for a complete discussion).
Molecular Cell
650
Holliday junctions bearing only a 3⬘ tail (Figure 2C) or
no tails at all (Figure 2D) were also tested in this assay.
With no 5⬘ single-stranded tail, DnaB cannot unwind
DNA and can only slowly load onto dsDNA. Thus, we
expect very little activity using DnaB with these two
substrates. Indeed, as the results of Figures 2C and
2D demonstrate, very little product accumulates using
these Holliday junction substrates.
The Holliday junction used in this assay contains heteroduplex arms; therefore, branch migration of this substrate requires the unwinding of two duplexes (1-2 and
3-4) without compensatory reannealing. This activity requires the input of energy. To confirm that DnaB-catalyzed branch migration is coupled to ATP hydrolysis,
we incubated the 5⬘-tailed Holliday junction with DnaB
and the nonhydrolyzable analog, AMP-PNP (Figure 2E).
There was no activity, as expected. Furthermore, the
activity profile of DnaB with various nucleotide cofactors
is similar to that previously published for unwinding (Figure 2F), suggesting a similar energy requirement (LeBowitz and McMacken, 1986). There was no activity when
DnaB was omitted from the reaction (data not shown).
If DnaB can drive branch migration of a synthetic
Holliday junction bearing a 5⬘ tail, it should split the
substrate into two, yielding the DNA products shown in
Reaction I of Figure 3A. However, DnaB is also a helicase, and these products are forked templates. Therefore, if DnaB should continue, in subsequent steps, to
reassociate with the products of Reaction I, it will unwind
them (shown in Reactions II and III of Figure 3A). Furthermore, reannealing can occur between products that
contain complementary sequences. Thus, many product species may be created if DnaB can drive branch
migration of a 5⬘-tailed Holliday junction substrate.
To determine if the reaction scheme illustrated in Figure 3A is the path taken by DnaB, we used several
approaches. First, we analyzed the time course of product accumulation; it matches that predicted by Figure
3A. The most abundant product to appear first in the
time course is composed of strands 1 and 4 (Figure 2A),
the predicted product of branch migration (Reaction I
of Figure 3A). Later, unannealed strand 1 accumulates,
the product of Reaction II. Still later, the species composed of strands 1 and 2 is visible. This species forms
upon reannealing of strand 1 (the product of Reaction
II in Figure 3A) with strand 2 (the product of Reaction
III in Figure 3A).
One may argue that the branch migration product (1-4
hybrid) is formed in two steps, direct unwinding of the
DNA strands, followed by reannealing. However, DnaB
cannot unwind a duplex bearing only a 5⬘ tail under
these conditions, as demonstrated in Figure 1. (The sequence of the strand bearing the 5⬘ tail is identical in
Figures 1B and 2A). Further, we measured the reannealing rate for strands 1 and 4 and determined the half-time
to be 60 min. In contrast, the half-time for reannealing
of strands 1 and 2 is 1.3 min. The large difference in
reannealing rates is explained by the ability of strand 4
to form an intramolecular stem-loop structure. Furthermore, if we melt the 5⬘ tail junction shown in Figure 2A
by heating to 95⬚C for 5 min and then allow reannealing
to occur at 37⬚C, the branch migration product composed of strands 1 and 4 is not seen as a reannealing
intermediate, whereas the 1-2 hybrid is readily detected
(data not shown).
To further support the conclusion that the reaction
proceeds according to the scheme in Figure 3A, Reaction II of this scheme was inhibited by reversing the
polarity of the bottom, or 3-4 duplex, of this junction
(Figure 3B). To reverse the polarity of the 3-4 duplex, a
5⬘-5⬘ DNA connection was incorporated into strand 4
(depicted as an open circle in Figure 3B), and a 3⬘-3⬘
connection was incorporated into strand 3 (depicted as
a closed circle in Figure 3B). These modifications result
in strand 4 having two 3⬘ ends, and strand 3 having two
5⬘ ends. A Holliday junction with these modifications is
similar to that in Figure 2A, except the polarity of the
3-4 duplex is reversed (Figure 3B).
The reversed polarity of the 3-4 duplex inhibits Reaction II of Figure 3A, because now the forked region of
the Reaction II substrate contains two 3⬘ tails, and DnaB
cannot unwind this substrate (Kaplan, 2000). With Reaction II inhibited, unannealed strand 1 cannot accumulate,
and neither can the species composed of strands 1 and
2. The gel in Figure 3B shows that these two products
do not accumulate, supporting the reaction scheme of
Figure 3A.
Next we designed a substrate to inhibit both Reaction
II and Reaction III, thereby leading to only the two primary products of branch migration in Reaction I of Figure 3A. We incorporated reverse polarity oligonucleotides into the 3-4 and 2-3 duplex regions of the Holliday
junction, as well as GC rich DNA into the 2-3 duplex
(Figure 3C). By making these modifications, the substrate of Reaction III of Figure 3A has a fork with two 5⬘
tails, but both strands change polarity at the duplex
junction. This inhibits, but does not completely block,
unwinding (Kaplan, 2000). Increasing the GC content of
the 2-3 duplex further inhibits unwinding of this substrate. As predicted by the scheme in Figure 3A, only
the branch migration product accumulates using this
substrate (Figure 3C).
As further proof that the reaction follows the scheme
of Figure 3A, we labeled a different strand of the 5⬘ tail
junction (strand 2) and analyzed the reaction of DnaB
with this Holliday junction (Figure 3D). Note that many
products are visible, but the first, most abundant product is composed of strands 2 and 3, as predicted by
Reaction I of Figure 3A. We then inhibited Reactions II
and III of Figure 3A as described above and found that
essentially only the branch migration product (composed of strands 2 and 3) is formed (Figure 3E).
T7 gp4B, but Not UvrD, Drives Branch Migration
of Holliday Junctions
Next, we studied other helicases to determine if the
branch migration activity discovered here for DnaB generalizes to other helicases. First, we examined the T7
gp4B helicase, a ring-shaped hexameric helicase that,
like DnaB, acts at a replication fork. We incubated gp4B
with dTTP and a 5⬘-tailed Holliday junction with reversed
polarity in duplexes 2-3 and 3-4 to inhibit Reactions II
and III of Figure 3A. As shown in Figure 4A, the Holliday
junction substrate is rapidly converted to the branch
migration product (1-4 hybrid). The reaction is nearly
complete by 1 min, faster than that observed for DnaB
DnaB Drives DNA Branch Migration
651
Figure 3. DnaB Catalyzes Branch Migration
of a Holliday Junction
(A) The scheme illustrates the proposed path
of product formation. First, DnaB, while encircling two strands, catalyzes branch migration
to provide duplexes 1-4 and 2-3 (Reaction I).
These products contain forked ends and can
be unwound by DnaB in subsequent steps,
as shown in Reactions II and III. Reannealing
of strands with complementary sequences
can also occur (not shown).
(B) DnaB action on a 5⬘-tailed Holliday junction with reversed polarity of the 3-4 duplex.
The open circle represents a 5⬘-5⬘ DNA connection, the closed circle represents a 3⬘-3⬘
connection.
(C) DnaB action on a 5⬘-tailed Holliday junction with reversed polarity of the 3-4 and 2-3
duplexes. The 2-3 duplex is GC rich.
(D) DnaB action on a 5⬘-tailed Holliday junction with strand 2 labeled with 32P.
(E) DnaB action on a 5⬘-tailed Holliday junction with strand 2 labeled with 32P. The 3-4
and 2-3 duplexes have reversed polarity. The
2-3 duplex is GC rich.
(Figure 3C). Thus, branch migration activity may be a
general property shared by members of this ring-shaped
helicase family.
We also examined UvrD, a helicase of the SF1 superfamily that does not form a ring-shape (Ali et al., 1999).
We used a helicase substrate that has a 3⬘ tail attached
to the Holliday junction, since UvrD unwinding is stimulated by a 3⬘ tail, not a 5⬘ tail (Figure 4B) (Matson, 1986).
As shown in Figure 4B, UvrD unwinds strand 4 of this
substrate, but no branch migration product (1-4 hybrid)
Figure 4. Hexameric T7 gp4B Also Catalyzes
Branch Migration, but UvrD Does Not
(A) Analysis of T7 gp4B helicase on a 5⬘-tailed
Holliday junction with reversed polarity of the
2-3 and 3-4 duplexes. The 2-3 duplex is GC
rich.
(B) Analysis of UvrD helicase with a 3⬘-tailed
Holliday junction with reversed polarity of the
2-3 duplex.
Molecular Cell
652
Figure 5. DnaB Encircles Both Strands 1 and
4 during Branch Migration, as Determined by
Biotin/Streptavidin Blocks
(A) Analysis of DnaB on a 5⬘-tailed Holliday
junction (strand 2 labeled) with the biotin/
streptavidin positioned on strand 4 or strand
1. Results of product analysis on a native gel
are quantified in the plot (squares, no biotin;
triangles, biotin on strand 1; circles, biotin on
strand 4).
(B) DnaB action on a 5⬘-tailed Holliday junction, with strand 1 labeled and the biotin/
streptavidin positioned on strand 3 or strand
2 (squares, no biotin; triangles, biotin on
strand 3, circles, biotin on strand 2).
is visible. Thus, branch migration activity is not a general
property of all helicases.
DnaB and T7 gp4B are likely acting in a processive
fashion during branch migration, since if these protein
rings were to dissociate from the Holliday junction during branch migration, the substrate would reanneal and
no activity would be observed. Further studies to follow
demonstrate that the DnaB ring surrounds the duplex
during branch migration, consistent with a processive
mechanism.
During Branch Migration, Strands 1 and 4, but Not 2
or 3, Pass through the Central Channel of DnaB
We next examined which DNA strands pass through the
central channel of DnaB during branch migration, using
biotin/streptavidin as a steric block. The diameter of
streptavidin is ⵑ45 Å, larger than the central channel of
DnaB helicase. Thus, streptavidin bound to a DNA
strand should prevent it from passing through the central
channel of DnaB, and movement should be blocked. In
contrast, when streptavidin is bound to the DNA strand
that passes on the outside of the DnaB ring, activity
should not be inhibited.
In the experiment of Figure 5, we replaced a dT with
a biotin-dT within the test strand and then added excess
streptavidin to the reaction to create a steric block. Biotin-dT still pairs with dA on the complementary strand
and has a spacer arm connecting biotin to dT. When
biotin-dT is present on strand 4 (Figure 5A), branch migration activity is abolished (open circles in the graph).
Likewise, when biotin-dT is present on strand 1, activity
is also completely blocked (open triangles in the graph
in Figure 5A). These data suggest that strands 1 and 4
of the Holliday junction pass through the central channel
of DnaB. (We previously showed that DnaB can displace
a small percentage of EBNA from duplex DNA in 4 min
[Figures 1D and 1E], but DnaB presumably does not
displace streptavidin here. Two likely explanations for
this are that streptavidin binds tighter to biotin than
EBNA to duplex DNA, and protein displacement during
branch migration may be slower compared to protein
displacement during translocation along duplex DNA.)
In contrast, when biotin-dT is present on strand 3
(Figure 5B), it has no effect on branch migration (open
triangles in the graph). When biotin-dT is present on
strand 2, there is a slight decrease in product accumulation at later time points (closed circles in the graph in
Figure 5B). This slight decrease may be due to streptavidin blocking Reaction III of Figure 3A. With Reaction
III inhibited, reannealing of the products of Reaction I
(Figure 3A) will occur faster, and less product will accumulate at later time points.
We conclude that the strand bearing the 5⬘ tail and
the strand that is complementary to it near the tail both
pass through the central channel of DnaB during branch
migration, whereas the other two strands pass outside
the DnaB ring.
As a control, we performed similar experiments with
the forked Holliday junction substrate, upon which DnaB
mainly acts as a helicase. Unwinding is inhibited only
by streptavidin bound to strand 1, as expected (see
Supplemental Figure S2 at http://www.molecule.org/
cgi/content/full/10/3/647/DC1).
During Branch Migration, DnaB Binds Only
to the Strand Bearing the 5ⴕ Tail
We next examined which strand DnaB actually binds to
during branch migration, using reverse polarity oligonucleotides (i.e., having internal 3⬘-3⬘ or 5⬘-5⬘ linkages). If
DnaB binds a particular strand during branch migration,
it should sense the chemistry and polarity of this strand,
and thus will not be able to move across a link that
reverses the strand polarity. In contrast, if DnaB does
not bind a particular strand, then its movement should
not be inhibited by a reversal in strand polarity.
In the experiments of Figure 6A, we constructed three
Holliday junction substrates that contain reverse polarity
linkages in either the 2-3, 3-4, or 1-2 duplex arms. Each
of these substrates was incubated with DnaB, and the
rate of product formation was compared to a reaction
using a substrate with no polarity reversal.
In our model, neither strand of the 2-3 duplex passes
through the central channel of DnaB. Since the DNA
binding site of DnaB is believed to be on the inside of
the ring, it seems unlikely that the protein binds to either
strand 2 or strand 3. Consistent with this idea, reversing
the polarity of the 2-3 duplex does not substantially
inhibit product accumulation (Figure 6A, open triangles).
(Since this polarity reversal inhibits Reaction III of Figure
3A, the reannealing rate to reform the Holliday junction
will increase, and less product will accumulate at later
time points.) In contrast, reversing the polarity of the 1-2
duplex completely abolished branch migration activity
(Figure 6A, open diamonds). Thus, DnaB likely makes
direct contact with one of these strands.
DnaB Drives DNA Branch Migration
653
Figure 6. DnaB Tracks on Only One Strand,
Even Though it Encircles Two Strands
DnaB action was analyzed on synthetic Holliday junctions with different modifications.
(A) Reaction rate of DnaB with 5⬘-tailed Holliday junctions containing a 2-3 duplex reverse
polarity (left diagram, triangles in graph), a
3-4 duplex reverse polarity (middle diagram,
circles in graph), a 1-2 duplex reverse polarity
(right diagram, diamonds in graph), or no reverse polarity (squares, no diagram shown).
(B) Reaction rate of DnaB with 5⬘-tailed Holliday junctions containing a shortened 1-2 duplex (left panel, triangles in graph) or a shortened 3-4 duplex (right panel, squares in
graph).
(C) Reaction rate of DnaB with 5⬘-tailed Holliday junctions containing no hexaethylene
glycol 1-phosphate (squares in graph), or
hexaethylene glycol 1-phosphate within
strand 2 (left panel, circles in graph) or strand
1 (right panel, triangles in graph). The zigzag
line represents replacement of nine nucleotides with three hexaethylene glycol 1-phosphate groups.
Reversing the polarity of the 3-4 duplex resulted in a
modest decrease in activity (Figure 4D, closed circles).
The 5⬘ to 5⬘ connection in strand 4 likely creates a steric
block as this region of DNA passes through the central
channel of DnaB. This effect may be combined with an
enhanced reannealing rate (Reaction II of Figure 3A is
blocked) to give the modest inhibition observed. If DnaB
were specifically binding to either strand of the 3-4 duplex during branch migration, one may expect to see a
much larger decrease in activity.
To confirm that the modest decrease observed for the
reverse polarity of the 3-4 duplex is not due to specific
binding of this duplex by DnaB, Holliday junction substrates with a short 3-4 duplex or a short 1-2 duplex
were tested. As expected, DnaB branch migration is
substantially inhibited by a shortened 1-2 duplex (open
triangles, Figure 6B). This result supports the conclusion
that DnaB binds to the 1-2 duplex during branch migration. In marked contrast, DnaB rapidly drives branch
migration of the duplex with a short 3-4 duplex (closed
squares, Figure 6B), supporting the idea that DnaB does
not bind to either strand of the 3-4 duplex.
The data for reversed polarity and shortened duplexes
demonstrate that DnaB binds to the 1-2 duplex, but
not the 2-3 duplex or the 3-4 duplex, during branch
migration. Which strand of the 1-2 duplex does DnaB
bind? Since strand 1, but not strand 2, of the 1-2 duplex
passes through the central channel of DnaB, it is likely
that strand 1 contacts DnaB. This idea is also consistent
with the ability of DnaB to translocate in the 5⬘ to 3⬘
direction along the strand it loads on.
To experimentally determine which strand of the 1-2
duplex DnaB binds, nine nucleotides within either strand
were replaced with three hexaethylene glycol 1-phosphate groups. This chemical group is too small to create
a steric block, but its chemistry is completely different
from DNA. Thus, if DnaB binds the strand with the hexaethylene glycol 1-phosphate, branch migration should
be blocked. If DnaB does not bind this strand, activity
should not be inhibited. As expected, hexaethylene glycol 1-phosphate within strand 1 (open triangles), but not
strand 2 (closed circles), substantially inhibited branch
migration (Figure 6C). Thus, DnaB binds to strand 1 but
not strand 2 during branch migration.
In conclusion, strands 1 and 4 pass through the central
channel of DnaB during branch migration, but DnaB
binds only to strand 1.
Discussion
This report reveals an activity of the ring-shaped hexameric helicase DnaB in which it actively translocates
along DNA with two strands positioned within the central
channel. This activity allows DnaB to displace proteins
Molecular Cell
654
Figure 7. DnaB Action in Branch Migration
and Protein Displacement
(A) Mechanism of DnaB branch migration of a
5⬘-tailed Holliday junction with heterologous
duplex arms. Step i: DnaB loads onto and
encircles strand 1. Step ii: DnaB slips onto
the 1-4 duplex with both strands positioned
in the central channel. Step iii: DnaB binds
mainly to strand 1 and translocates along this
strand in the 5⬘ to 3⬘ direction with enough
force to simultaneously unwind the 1-2 and
3-4 duplexes. Step iv: once branch migration
is complete, DnaB dissociates.
(B) DnaB clears protein upstream from a leading strand nick (left panel) or a lagging strand
lesion (right panel). Left panel: when DnaB
encounters a nick in the leading strand, it may
encircle both parental strands and translocate upstream. DnaB then displaces DNAbound proteins, which may enable DNA repair proteins to initiate repair. Right panel:
DnaB may bind to the lagging strand downstream from a polymerase stalled at a lesion.
DnaB will translocate upstream and displace
the DNA polymerase, thereby enabling proteins to repair the lesion.
(C) DnaB-catalyzed branch migration near a
replication fork. Left panel: in gap repair, the
strand bearing the DNA lesion is paired with
a sister strand via recombination (left branch
of pathway) or fork regression (right branch
of pathway). In the recombinative pathway,
DnaB may bind to the leading strand and
drive branch migration of the Holliday junction away from the replication fork, thereby
allowing DNA repair. In fork regression, DnaB
may bind to the lagging strand and drive DNA
branch migration, thereby moving the DNA
lesion away from the replication fork to allow
repair. Right panel: in daughter-strand gap
repair, DnaB may bind to the leading strand
and drive branch migration of the Holliday
junction away from the replication fork.
that are tightly bound to the DNA, without unwinding
the DNA. We have also demonstrated that DnaB can
drive branch migration of a Holliday junction. Neither
of these activities has been observed previously for a
replication fork helicase. This report also demonstrates
that the T7 gp4B helicase can drive branch migration,
suggesting that the activity described for DnaB is general for this family of helicases. Upon study of the mechanism of DnaB-catalyzed branch migration, we found
that two strands pass through the central channel of
DnaB during branch migration, but DnaB binds to only
one of these strands.
Model of How DnaB Drives Branch Migration
of a Holliday Junction
Combining the results herein with other studies of DnaB,
we propose a model for DnaB branch migration in Figure
7A. DnaB is known to load onto substrate DNA by encircling a 5⬘ ssDNA tail (Bujalowski and Jezewwska, 2000).
In Figure 7A, this 5⬘ tail is strand 1 of the Holliday junction
(panel i). DnaB tracks along the ssDNA in the 5⬘ to 3⬘
direction (LeBowitz and McMacken, 1986). Upon encountering the first complementary strand (strand 4),
DnaB continues to track along the same strand, but now
both strands (strands 4 and 1) are positioned within the
central channel of the protein (panel ii). When DnaB
reaches the four-way junction, it continues to actively
translocate along strand 1 in the 5⬘ to 3⬘ direction and
essentially rips the top and bottom arms of the Holliday
junction apart (panel iii). DnaB binds only to strand 1
during this branch migration process. During this action,
strand 1 is unwound from strand 2, and strand 4 is
concomitantly unwound from strand 3. Furthermore,
strands 1 and 4 are both within the central channel of
the protein, but they are not annealed to each other,
since they are not complementary in this region. Once
DnaB completes unwinding of both duplexes, the enzyme dissociates (panel iv).
DnaB tracks along strand 1, and thus it is easy to
imagine it being unwound from strand 2 by DnaB during
branch migration. However, this study indicates that
DnaB encircles strand 4 but does not bind it tightly for
DNA tracking. Therefore, it is less easy to understand
how strand 4 is unwound from strand 3, since DnaB
does not contact either strand. We propose that as DnaB
translocates along strand 1 and pulls it through the central channel, strand 4 will be pulled indirectly by DnaB
DnaB Drives DNA Branch Migration
655
due to the fact that strand 4 is paired to strand 1. This
indirect pulling of strand 4 through the central channel,
with strand 3 positioned outside the protein ring, forces
the unwinding of strand 4 from strand 3.
Unified Model of DnaB Activity
It is possible that DnaB has up to four modes of active
translocation: translocation along single-stranded DNA,
unwinding, translocation along double-stranded DNA to
dislodge proteins, and branch migration. However, it is
likely that all of these processes occur by the same
mechanism. In previous work, it was described how
translocation along single-stranded DNA and unwinding
occur by the same mechanism; namely, translocation
along single-stranded DNA accomplishes unwinding if
the second strand is positioned on the outside of the
protein ring, even though DnaB does not contact this
second strand (Kaplan, 2000).
When DnaB drives branch migration of a Holliday junction with heterologous duplex arms, it also translocates
along one strand of DNA in the 5⬘ to 3⬘ direction (Figure
7A). The force generated during this translocation is
capable of simultaneously unwinding two DNA duplexes, even though the protein is binding to just one
DNA strand. It is the fact that the second strand is also
positioned within the central channel that results in
branch migration, even though DnaB is otherwise functioning as an unwinding protein. Likewise, protein bound
to DNA will not fit through the center of DnaB and may
simply be driven off the duplex as DnaB translocates
along it. Thus, unwinding, branch migration, and protein
displacement may all be catalyzed by the same mechanism.
Why Does DnaB Have a Ring Shape?
DnaB translocates for thousands of base pairs during
replication, and a ring shape may have evolved to increase its processivity. However, the ring shape may
also be instrumental for DnaB to catalyze unwinding
and branch migration. A helicase separates two complementary strands of DNA. By having one strand passing
through the central channel and one strand passing
outside the central channel, the strands are physically
separated from each other. Physical separation inhibits
reannealing of the complementary strands, thereby assisting the unwinding reaction.
During branch migration, two duplexes are simultaneously unwound. For either duplex, one strand is located
within the central channel, while its complementary
strand is located outside (Figure 7A). Thus, the helicase
ring allows for physical separation of two strands of DNA
from their complementary partners, thereby inhibiting
reannealing of both duplexes. Thus, a protein’s ring
shape may help it accomplish branch migration.
DNA unwinding and branch migration can be catalyzed by proteins that do not form ring shapes. For these
proteins, other mechanisms may be used to separate
the strands and ensure that once the strands are unwound, they do not immediately reanneal.
Comparison to Other E. coli Proteins that Drive
Branch Migration of Holliday Junctions
There are several other E. coli proteins that drive branch
migration of Holliday junctions, including RecG and
RuvAB proteins. RecG is not hexameric and does not
form a ring (Singleton et al., 2001). RuvAB, on the other
hand, is hexameric, forms a ring shape, and has many
biochemical activities that are shared with DnaB (for a
review, see West, 1997). As with DnaB, the RuvAB ring
can encircle two strands of DNA. Moreover, RuvAB unwinds DNA with 5⬘ to 3⬘ polarity (Tsaneva et al., 1993).
One difference between RuvAB and DnaB is the way
the proteins load onto DNA. DnaB loads onto a 5⬘ singlestranded tail and then migrates toward the Holliday junction. RuvA binds directly to the four-way junction and
recruits RuvB to that site (Parsons and West, 1993). A
second difference is that DnaB provides both strand
separation and ATP-driven motor activities, whereas
RuvA has an acidic pin to achieve strand separation
(Ariyoshi et al., 2000), while RuvB provides the ATPmotor function (Yamada et al., 2001).
Implications for Collision of DnaB with Other DNA
Binding Proteins In Vivo
In vitro work shows that the T4 gp41 protein can displace
a biotin-streptavidin complex from single-stranded DNA
(Morris and Raney, 1999). This suggests that when a
replication fork helicase translocates along singlestrand DNA, it can exert force and dislodge proteins
from its path. Consistent with this idea, it was shown
that T4 gp41, while unwinding DNA at a replication fork,
can displace RNA polymerase when the protein is transcribing on the lagging strand (Liu and Alberts, 1995),
but not the leading strand.
We have now shown that when DnaB is translocating
along DNA with two strands positioned in the central
channel, it can also displace proteins. In this case, the
DNA is not concomitantly unwound. This task may become important if DnaB was unwinding DNA at a replication fork and encountered a nick in the leading strand,
in which case both strands of DNA would enter the
central channel (Figure 7B, left panel). This event could
accomplish several things at once: (1) stop DNA unwinding, (2) allow DnaB to strip the broken DNA end of proteins in preparation for repair, and (3) retain DnaB on
DNA as a participant in the ensuing repair process.
DnaB-catalyzed protein displacement may be important in other cellular processes. For example, DnaB may
load onto the lagging strand downstream from a stalled
DNA polymerase and then translocate upstream, dislodging the polymerase to allow proteins to repair the
lesion (Figure 7B, right panel). In addition to DnaB, other
helicases or translocases may be involved in protein
displacement in vivo. Using nucleosome templates as
a model substrate for protein-bound DNA, the E. coli
RecBCD and RuvAB proteins have been shown to displace nucleosomes as they unwind DNA (Eggleston et
al., 1995; Grigoriev and Hsieh, 1998).
DnaB May Be Directly Involved in DNA
Recombination In Vivo
When a replication fork encounters a DNA lesion, a recombinative repair process may be initiated to repair the
lesion and restart replication. The two major pathways of
recombinative repair include double-strand break repair, if the DNA lesion is a nick, and daughter-strand
gap repair, if the lesion is not a nick. In both of these
Molecular Cell
656
recombinative repair processes, a Holliday junction is a
likely DNA intermediate (for a review, see Cox, 2000).
The RuvAB and/or RecG proteins have previously been
ascribed the role of driving branch migration of these
Holliday junctions, but one can now consider that DnaB
may catalyze this reaction (Figure 7C). Furthermore,
when a replication fork stalls, it may reverse or regress.
This process, which may be meditated by RecG
(McGlynn et al., 2001), RecA (Robu et al., 2001; Seigneur,
2000), or by positive torsional strain of the genomic
DNA (Postow et al., 2001), creates a Holliday junction
structure. DnaB may drive branch migration of this Holliday junction as well (Figure 7C).
Data with temperature sensitive mutations implicate
a role of DnaB in recombination. For example, repeated
genes and sequences are prone to genetic rearrangements, including deletions. These deletions are markedly enhanced in dnaB mutant strains in a manner that is
mostly recA and lexA-dependent (Saveson and Lovett,
1997). Similarly, precise excision of transposon Tn10 is
enhanced by a mutation in the dnaB gene (Nagel and
Chan, 2000), and expansion of DNA repeats in E. coli is
also stimulated by mutations in dnaB (Morag et al.,
1999). Furthermore, overproduction of DnaB in E. coli
induces illegitimate recombination between short regions of homology (Yamashita et al., 1999). Previous
explanations for the phenotypes described above focused upon the role of DnaB as a replication fork helicase. In light of the data presented in this report, one
may now consider that these phenotypes could be attributed to a direct role of DnaB in recombination.
Experimental Procedures
Proteins and DNA
Proteins were expressed in E. coli and purified as described: DnaB
(Yuzhakov et al., 1996), EBNA1PK (Kelman et al., 1995b), and UvrD
(Runyan et al., 1993). pGEMoriP plasmid contains 24 EBNA1 binding
sites (Frappier and O’Donnell, 1991). DNA oligonucleotides are listed
in Supplemental Table S1 at http://www.molecule.org/cgi/content/
full/10/3/647/DC1. Oligonucleotides were labeled with 32P as previously described (Kaplan, 2000).
Unwinding and Holliday Junction Assays
All manipulations were performed in microfuge tubes on ice unless
otherwise stated.
Oligonucleotides were annealed by mixing the 32P-labeled strand
(100 nM) with unlabeled strands in 36 mM Tris-HCl, 17 mM magnesium acetate, 34% glycerol, 230 ␮M EDTA, 67 ␮g/ml BSA, 8.3 mM
DTT (pH 7.5) at 37⬚C overnight. Oligonucleotides complementary
to the labeled strand were at 150 nM, and oligonucleotides not
complementary to the labeled strand were 225 nM.
Enzyme reactions were incubated at 37⬚C for the time indicated
and contained (unless otherwise stated) 1 nM DNA substrate (concentration of labeled strand), 500 nM E. coli DnaB, 5 mM ATP, 5
mM creatine phosphate, 20 ␮g/ml creatine kinase, 20 mM Tris-HCl,
10 mM magnesium acetate, 20% glycerol, 100 ␮M EDTA, 40 ␮g/ml
BSA, and 5 mM DTT (pH 7.5) in a final volume of 20 ␮l. Reactions
were quenched by adding 1 ␮l of Proteinase K (10 mg/ml) and
incubating for 1 min. 5 ␮l 2% SDS and 80 mM EDTA, followed by
5 ␮l 15% Ficoll, 0.25% xylene cyanol FF, were then added.
Assays containing T7 gp4B or UvrD were identical to those using
DnaB, except that DnaB was replaced with either 500 nM T7 gp4B
or 50 nM UvrD. For T7 gp4B, 5 mM dTTP replaced ATP. 50 nM
streptavidin was preequilibrated with DNA before adding to the
assay mixture.
DNA products were analyzed by 8% or 12% PAGE using 1⫻ TBE
(90 mM Tris-HCl-Borate, 2 mM EDTA), 175 V, and 25⬚C.
Gels were dried and then exposed to a Phospor-imaging screen
(Molecular Dynamics). For the unwinding reaction, product is defined as single-stranded DNA. For Holliday junction reactions, products are defined by any species that migrates faster than substrate.
The percentage of products is calculated as follows: % products ⫽
(%Ps ⫺ %P0)/(1 ⫺ %P0), where %Ps is the percent products in the
sample lane, and %P0 is the percent products in the unreacted
substrate.
EBNA1 Displacement Assay
DNA was annealed as described above, except DNA strands were
unlabeled and each was at 100 nM. EBNA1PK was labeled with
[␥-32P]ATP using cAMP-dependent protein kinase as described (Kelman et al., 1995a). 32P-EBNA1PK was prebound to DNA substrate by
mixing 20 ␮l of 32P-EBNA1PK (307 nM stock, 40,800 cpm/␮l) with
100 ␮l of DNA oligonucleotide duplex (200 nM stock), followed by
incubation at 25⬚C for 30 min.
Reactions contained (unless otherwise stated) 40 nM DNA oligonucleotide duplex, 12 nM 32P-EBNA1PK, 160 nM pGEMoriP, 500 nM
E. coli DnaB, 5 mM ATP, 5 mM creatine phosphate, 20 ␮g/ml creatine
kinase, 20 mM Tris-HCl, 10 mM magnesium acetate, 20% glycerol,
100 ␮M EDTA, 40 ␮g/ml BSA, and 5 mM DTT (pH 8.0). Reactions
were incubated at 37⬚C for the indicated times, and then 6.5 ␮l of
100 mM Tris-HCl, 100 mM EDTA, 15% Ficoll, and 0.25% xylene
cyanol FF (pH 7.5) were added. Reactions were analyzed by a 0.7%
agarose gel using 1⫻ TBE, 100 V, and 25⬚C. The gel was dried and
then analyzed as described above.
Acknowledgments
We thank Dr. Smita Patel for supplying the T7 gp4B protein and
Dr. Susan Taylor for supplying the catalytic subunit of the cAMPdependent protein kinase. We thank Dr. Benedicte Michel and Dr.
Justin Courcelle for critical reading of this manuscript. Thanks to
everyone in the O’Donnell Lab, especially Dr. Monett Librizi for purification of DnaB, Dr. Francisco Lopes de Saro for purification of UvrD,
Dr. Dan Zhang for purification of EBNA1PK, and Dr. Irina Bruck for
suggestions throughout this project. This research was supported
by grant GM 62540 from the NIH and by HHMI. D.L.K. is the Leon and
Toby Cooperman Fellow of the Damon Runyan Cancer Research
Foundation (DRG #1663).
Received: May 17, 2002
Revised: July 26, 2002
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