The N-terminal domain of Escherichia coli RecA have multiple functions in promoting homologous recombination.

Abstract

Escherichia coli RecA mediates homologous recombination, a process essential to maintaining genome integrity. In the presence of ATP, RecA proteins bind a single-stranded DNA (ssDNA) to form a RecA-ssDNA presynaptic nucleoprotein filament that captures donor double-stranded DNA (dsDNA), searches for homology, and then catalyzes the strand exchange between ssDNA and dsDNA to produce a new heteroduplex DNA. Based upon a recently reported crystal structure of the RecA-ssDNA nucleoprotein filament, we carried out structural and functional studies of the N-terminal domain (NTD) of the RecA protein. The RecA NTD was thought to be required for monomer-monomer interaction. Here we report that it has two other distinct roles in promoting homologous recombination. It first facilitates the formation of a RecA-ssDNA presynaptic nucleoprotein filament by converting ATP to an ADP-Pi intermediate. Then, once the RecA-ssDNA presynaptic nucleoprotein filament is stably assembled in the presence of ATPgammaS, the NTD is required to capture donor dsDNA. Our results also suggest that the second function of NTD may be similar to that of Arg243 and Lys245, which were implicated earlier as binding sites of donor dsDNA. A two-step model is proposed to explain how a RecA-ssDNA presynaptic nucleoprotein filament interacts with donor dsDNA.

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BioMed Central
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Journal of Biomedical Science
Open Access
Research
The N-terminal domain of Escherichia coli RecA have multiple
functions in promoting homologous recombination
Chien-Der Lee and Ting-Fang Wang*
Address: Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan
Email: Chien-Der Lee - cdlee@gate.sincia.edu.tw; Ting-Fang Wang* - tfwang@gate.sinica.edu.tw
* Corresponding author
Abstract
Escherichia coli RecA mediates homologous recombination, a process essential to maintaining
genome integrity. In the presence of ATP, RecA proteins bind a single-stranded DNA (ssDNA) to
form a RecA-ssDNA presynaptic nucleoprotein filament that captures donor double-stranded
DNA (dsDNA), searches for homology, and then catalyzes the strand exchange between ssDNA
and dsDNA to produce a new heteroduplex DNA. Based upon a recently reported crystal
structure of the RecA-ssDNA nucleoprotein filament, we carried out structural and functional
studies of the N-terminal domain (NTD) of the RecA protein. The RecA NTD was thought to be
required for monomer-monomer interaction. Here we report that it has two other distinct roles
in promoting homologous recombination. It first facilitates the formation of a RecA-ssDNA
presynaptic nucleoprotein filament by converting ATP to an ADP-Pi intermediate. Then, once the
RecA-ssDNA presynaptic nucleoprotein filament is stably assembled in the presence of ATPS, the
NTD is required to capture donor dsDNA. Our results also suggest that the second function of
NTD may be similar to that of Arg243 and Lys245, which were implicated earlier as binding sites
of donor dsDNA. A two-step model is proposed to explain how a RecA-ssDNA presynaptic
nucleoprotein filament interacts with donor dsDNA.
Background
Escherichia coli RecA is the founding member of the RecA
protein family. It is essential for the initiation of repair of
DNA breaks via homologous recombination, induction of
the DNA damage-induced 'SOS' response, and activation
of translesion DNA synthesis, as well as development and
transmission of antibiotic resistance genes [1,2]. Nearly
all known functions of RecA require the formation of a
presynaptic helical filament comprised of single-stranded
DNA (ssDNA) bound to multiple RecA monomers with
ATP. During homologous recombination, this activated
form of the helical filament is capable of interacting with
homologous double-stranded DNA (dsDNA) to form a
heteroduplex DNA molecule. Eventually, the DNA strands
are exchanged, resulting in the displacement of one of the
original duplex strands and the subsequent creation of a
new heteroduplex (or D-loop). This function is evolution-
arily conserved in other members of the RecA family,
including archaeal RadA and the eukaryotic proteins,
Rad51 and Dmc1.
The RecA monomer has three major structural domains: a
small N-terminal domain (NTD), a core ATPase domain
(CAD), and a large C-terminal domain (CTD). By con-
trast, the monomers of RadA/Rad51/Dmc1 consist of a
CAD and a larger NTD. The CAD, often referred to as the
Published: 1 April 2009
Journal of Biomedical Science 2009, 16:37doi:10.1186/1423-0127-16-37
Received: 18 February 2009
Accepted: 1 April 2009
This article is available from: http://www.jbiomedsci.com/content/16/1/37
© 2009 Lee and Wang; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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RecA fold [3], is structurally similar to the ATPase
domains of DNA/RNA helicases, F1 ATPases, chaperone-
like ATPases, and membrane transporters [4]. Highly con-
served in all RecA family proteins, the CAD contains two
disordered loops (denoted the L1 and L2 motifs) that
bind to ssDNA and are responsible for ssDNA-stimulated
ATPase activity [5]. All RecA family proteins are polymer-
ized via a polymerization motif located between the NTD
and the CAD. The polymerization motif contains a hydro-
phobic residue (isoleucine 26 in E. coli RecA; phenyla-
lanines in RadA, Rad51, and Dmc1) that docks within the
hydrophobic pocket of the neighboring CAD. This inter-
action was also observed at the binding interface between
a human Rad51 monomer and a BRC repeat of BRCA2
tumor suppressor protein [6-9]. The polymerization motif
of archaeal RadA protein is responsible for the assembly
of different quaternary structures, including toroidal
rings, as well as right-handed and left-handed helical fila-
ments [10,11].
The crystal structures of RecA-ssDNA and RecA-dsDNA
nucleoprotein complexes with ADP-AlF4--Mg2+ have
recently been reported [12]. These new structures have
provided unprecedented new insights into the mecha-
nisms and energetics of RecA protein. In the RecA-ssDNA-
ADP-AlF4--Mg2+ filament complex, the ssDNA is bound by
the L1 and L2 loop regions as well as by the N-terminal
portion of the F and G helices that follow L1 and L2,
respectively. The ADP-AlF4--Mg2+ is sandwiched between
the CADs of two adjacent RecA protomers in a completely
buried environment. AlF4- group is coordinated by the
side chains of Lys248 and Lys250. Lys250 also hydrogen
bonds to the side chain of Glu96 in the neighboring RecA
protomer. Glu96 is the catalytic residue thought to acti-
vate a water molecule for nucleophilic attack on the -
phosphate. This second interface is absent in the inactive
filament, where the corresponding interface of the ATP
analogue, adenylyl-imidodiphosphate (AMP-PNP), is sol-
vent exposed. Therefore, the charged-stabilized hydrogen
bonds that Lys248 and Lys250 make to the AlF4- group
could explain the ATP-dependency of DNA binding,
because the -phosphate of ATP is sensed across the RecA-
RecA interface cooperating with DNA binding to promote
the transition to the active filament state. Close to the fil-
ament axis, the ssDNA is extended 50% in length relative
to B-form DNA with the same sequence. Each RecA mon-
omer interacts with three nucleotides of the DNA (a tri-
plet), and each triplet is also bound by three contiguous
RecA monomers. Strikingly, the DNA-RecA interaction, or
DNA extension, is not isotropic at the nucleotide level;
instead, the DNA comprises a three nucleotide segment
with a nearly normal B-form distance between bases (an
axial rise of 3.5–4.2 Å for ssDNA and 3.2–4.5 Å for
dsDNA), followed by a long, untwisted internucleotide
stretch (~7.1–7.8 Å in ssDNA and 8.4 Å in ssDNA) before
the next nucleotide triplet, in a repeating pattern. Such an
unusual repeat pattern of DNA extension reveals a new
structural basis of the dynamics of filament assembly in
the presence of ssDNA, including initiation (or nuclea-
tion) of filament assembly and the observed cooperativity
of RecA-DNA binding.
The RecA-dsDNA-ADP-AlF4--Mg2+ crystal structure was
postulated to be an end product after the strand exchange
reaction between a RecA-ssDNA nucleoprotein filament
and a homologous dsDNA target [12,13], implying that
RecA protein filaments may complete all functions
(including ssDNA binding, donor dsDNA capturing, and
strand exchange) in right-handed forms and also within
the filament axes. Here, we considered an alternative pos-
sibility: that the RecA-dsDNA crystal structures might sim-
ply represent annealing products of the ssDNA in the
RecA-ssDNA nucleoprotein filament and a complemen-
tary ssDNA. First, in the RecA-ssDNA filament structure,
the purine and pyrimidine bases of bound ssDNA are out-
wardly exposed. Second, the complementary ssDNA in
the RecA-dsDNA structure makes very few physical con-
tacts with the RecA protein filament, indicating that the
annealing of these two ssDNAs has very little impact on
the protein structures. Indeed, the overall protein struc-
tures of RecA-dsDNA filaments are highly similar to those
of RecA-ssDNA-ADP-AlF4--Mg2+ structures
Because the molecular mechanism of the strand exchange
reaction is still not understood, it is important to examine
these two different possibilities further.
[12,13].
The RecA-ssDNA crystal structures also indicate that
Arg243 and Lys245 might constitute a binding site for the
donor dsDNA during the strand exchange reaction.
Arg243 and Lys245 are ~25 Å away from the filament axis
and have a repeat distance of ~28 Å along the filament
axis. Their positively charged side chains are solvent-
exposed and face towards the central axis of the RecA-
ssDNA helical filament (Figure 1A) [12]. This is consistent
with previous reports that these positively charged resi-
dues are responsible for dsDNA capture during homology
pairing and strand exchange [14,15]. However, a closer
look at the RecA-ssDNA crystal structure revealed that the
positively charged side chains of Arg243 and Lys245 are
not exposed to the exterior surface of the nucleoprotein
filament; they reside inside the filament (Figure 1B). We
speculated that these two amino acid residues might not
be solely responsible for dsDNA recruitment. Other struc-
tural element(s), located at the outermost surface of the
RecA-ssDNA presynaptic filament, may assist the RecA-
ssDNA presynaptic filament in its search for donor
dsDNA.
In the present study, we report that the NTD of RecA may
have such a function. Additional mutant analysis revealed

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that it might have at least two distinct roles in promoting
the RecA-mediated homologous recombination reaction.
Materials and methods
RecA protein production, enzymatic assays, and DNA
substrates
An improved SUMO fusion protein expression system
[16] was used to rapidly produce RecA proteins in
The nuclease assay, D-loop formation assay, ssDNA-
dependent ATPase activity assay, and DNA substrates used
.
in this study have also been described in detail [16].
ATPS (Adenosine 5'-O-(3-thio)triphosphate) and AMP-
PNP were purchase from Sigma Aldrich.
Electron microscopy
The wild-type and mutant
were incubated with 4 M circular X174 dsDNA (in base
pairs [bps]) at 37°C for 30 min in reaction buffer (1 mM
ATPS, 10 mM Mg2+-acetate, 100 mM Na acetate, 25 mM
Tris-Cl pH 7.4), and were chilled on ice to stop the reac-
RecA proteins (2 M)
Structure of a RecA-ssDNA-ADP-AlF4--Mg2+ presynaptic nucleoprotein filament
Figure 1
Structure of a RecA-ssDNA-ADP-AlF4--Mg2+ presynaptic nucleoprotein filament. Shown are ribbon diagrams of
side (A) and top (B) views of the recently reported 3CMU crystal structure [12]. The NTDs and ssDNA are shown in yellow
and red, respectively. The side chains of three charged residues are depicted as a ball-and-stick model. (C) Surface charge
potential of the 3CMU RecA-ssDNA-ADP-AlF4--Mg2+ presynaptic nucleoprotein filament. The positively and negatively charged
regions are indicated in blue and red, respectively. The positions of Lys8 [1], Lys19 [2], and Lys23 [3] are indicated.

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tion. A droplet (4 l) was placed on a copper grid (300
mesh, Pelco, USA) coated with fresh carbon for 1 min at
room temperature. Excess buffer was then carefully blot-
ted away from the edge of the grid with Whatman #1 filter
paper (Whatman Inc., USA). After staining for 4 min with
2.5% uranyl acetate, excess liquid was removed and the
samples were air dried at room temperature. Bio-transmis-
sion EM was performed with a Tecnai G2 Spirit Bio TWIN
(FEI Co., Netherlands) using an acceleration voltage of
120 kV. Images were recorded with a slowscan CCD cam-
era (Gatan MultiScan 600) at a resolution of at least 1024
× 1024 pixels.
Duplex DNA capture assay
The duplex DNA capture assay was carried out using a pro-
tocol modified from that described previously for Rad51
and Hop2-Mnd1 proteins [17]. To obtain presynaptic fil-
aments for the dsDNA capture assay, a 5'-biotinylated
ssDNA PA1656 (15 M in nucleotides) and RecA proteins
(5 or 20 M) were mixed with 4 L streptavidin-coated
magnetic beads (Novagen, USA) in 20 L of buffer C (20
mM HEPES-KOH at pH 7.0, 1 mM DTT, 100 ng/mL BSA,
2 mM Mg2+-acetate, 5% glycerol and 1 mM ATPS) for 5
min at 37°C. BSA was used as a negative control for RecA-
DNA binding reactions. The magnetic beads were isolated
with a magnetic separator (Novagen). The supernatant
(denoted as "S1") was set aside for later analysis. The mag-
netic beads were washed twice with 20 mL buffer C, and
then mixed with 15 M of donor dsDNA (bps) in 20 L of
buffer D (20 mM HEPES-KOH at pH 7.0, 1 mM DTT, 11
mM Mg2+-acetate, 5% glycerol) at 37°C for 10 min with
gentle mixing every 1 min. This donor dsDNA was 300
bps in length, and its central region was homologous to
PA1656. The magnetic beads were isolated again with a
magnetic separator. The supernatant (denoted as "S2")
was set aside for later analysis. After the magnetic beads
were washed twice with 20 L of buffer E (20 mM HEPES-
KOH at pH 7.0, 1 mM DTT, 11 mM Mg2+-acetate, 5% glyc-
erol and 1 mM ATPS), proteins and DNA substrates were
eluted by incubating with 20 mL of 1% SDS. The SDS elu-
ates (denoted as "B") were separated on either a 12% SDS-
PAGE stained with Coomassie blue to visualize bovine
serum albumin (BSA) and RecA proteins, or a 1.5% agar-
ose gel stained with ethidium bromide to visualize the
donor dsDNA.
Results
The NTD of RecA is similar in amino acid sequence to a
functional motif of RadA/Rad51/Dmc1
The NTD of RecA contains only 33 amino acid res-
idues and is much smaller in size than the RadA/Rad51/
Dmc1 NTDs (> 60 amino acids). The crystal structure of
the RecA-ssDNA presynaptic filament reveals that NTDs
are located at the exterior surface of the helical filament.
Moreover, NTDs are not directly involved in RecA-ssDNA
interaction or RecA-ATP binding (Figure 1A) [12]. A
superimposition of RecA-RecA pairs from the active RecA-
ssDNA presynaptic filament onto an inactive RecA protein
filament (achieved by aligning the CADs) revealed a large
conformational change in the hinge region (residues 31–
40) that connects the NTD and CAD. As a result, these two
filaments differ by a 32° rotation and an 18.5 Å transla-
tion of the CAD [12]. This hinge region is located imme-
diately after the polymerization motif (
RecA), which functions as a fulcrum to mediate a large
conformational change in response to ssDNA binding
[12]. A similar scenario was also reported in a structural
transition of the RecA protein filament from a compressed
form to a relaxed form [10]. Intriguingly, RadA/Rad51/
Dmc1 protein also contains a similar structural motif that
we referred to previously as the subunit rotation motif
(SRM) [18]. The SRM also uses the polymerization motif
as a fulcrum to mediate rotation along the central axis of
the archaeal RadA protein polymer. Continuous clock-
wise axial rotation of archaeal RadA proteins is responsi-
ble for the progression of stepwise structural transitions:
first from an inactive ring to a right-handed filament with
6 monomers per helical pitch, then to an overextended
right-handed filament with 3 monomers per helical pitch,
and, finally, to a left-handed filament with 4 monomers
per helical pitch[10,11,18]. A key consequence of these
structural transitions is the progressive relocation of the
NTDs and the L1 ssDNA binding motif. The NTD has
been shown to mediate donor dsDNA binding in both
human Rad51 [19] and archaeal RadA [18]. In the RadA
right-handed helical filament with 6 monomers per heli-
cal pitch, L1 resides near the axis filament and the NTD is
located at the exterior surface of the filament. In contrast,
in the overextended right-handed helical filament with 3
monomers per helical pitch, L1 relocates to the exterior
surface of the filament and constitutes an outward-open-
ing palm structure in combination with the NTD. Inside
this palm structure, 5 conserved basic amino acid residues
(Lys27 and Lys60 of the NTD; and Arg117, Arg223, and
Arg229 of the L1 motif) of Sulfolobus solfataricus RadA
(SsoRadA) surround this pocket (~25 Å in diameter),
which may be wide enough to simultaneously accommo-
date an ssDNA and a donor dsDNA [18]. All five posi-
tively charged residues are evolutionarily conserved in all
archaeal and eukaryotic RecA family proteins. Therefore,
the overextended right-handed filament structure of Sso-
RadA was proposed to represent a structural intermediate
during the homologous search and pairing process of
archaeal and eukaryotic RecA family proteins [18]. Simi-
larly, as described above, the RecA-ssDNA-ADP-AlF4--
Mg2+ nucleoprotein filament was also overextended
[12,13].
., Ile26 of
The NTD of RecA was previously considered to be struc-
turally and functionally distinct from those of Rad51/

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Dmc1/RadA proteins. The RecA NTD contains only a helix
(residues 1–23) and a -loop (residues 24–33). By con-
trast, the NTDs of archaeal and eukaryotic RecA family
proteins are composed of two helix-hairpin-helix (HhH)
motifs, in which a pseudo two-fold unit is composed of
two HhH motifs linked by a connector -helix. The HhH
motifs and the connector -helix are denoted as H1'h'H2',
H1hH2, and Hc. Each HhH motif contains two helices
(denoted as H1, H1', H2, or H2') and a hairpin (denoted
as h or h') [18]. We reported earlier that the second
H1hH2 motif of SsoRadA (i.e., the 3-3-4 region indi-
cated in Figure 2A) is located at the outer surface of the
NTD and constitutes a positively charged patch that is
responsible for dsDNA binding. Intriguingly, we found
that the -helix of the RecA NTD (residues 1–23) shows
significant amino acid sequence conservation with the
second H1hH2 motif of archaeal and eukaryotic RecA
family proteins. First, Gly15 and Lys23 of RecA are con-
The E. coli RecA NTD exhibits significant amino acid sequence homology with the NTDs of the homologous proteins Rad51/Dmc1/RadA.
Figure 2
The E. coli RecA NTD exhibits significant amino acid sequence homology with the NTDs of the homologous
proteins Rad51/Dmc1/RadA. (A) Sequence alignment of the NTDs of RecA proteins from S. solfataricus (SsoRadA), M. vol-
tae (MvRadA), P. furiosus (PfRad51), H. sapiens (HsRad51 and HsDmc1), S. cerevisiae (ScDmc1 and ScRad51), and E. coli (EcRecA).
Secondary structural features of the left-handed SsoRadA helical filament are indicated in cyan (-helices)[10]. Functional
motifs are indicated under their corresponding amino acid sequences: the first HhH motif (H'h'H2'), core helix (Hc the), and
the second HhH motif (H1Hh2). (B) The NTD carries out a large conformational change in response to ssDNA and ATPS.
Shown are ribbon diagrams of the monomeric RecA structures in the RecA-ssDNA-ADP-AlF4- presynaptic filament [12] and in
the inactive RecA protein helical filament [3]. The ssDNA and RecA polypeptides are shown in gold and green, respectively.
The side chains of Glu18, Lys23, and Arg33 are depicted as ball-and-stick models.