Characterization of the DNA-unwinding Activity of Human RECQ1, a
Helicase Specifically Stimulated by Human Replication Protein A*
Sheng
Cui,
Raffaella
Klima,
Alex
Ochem,
Daniele
Arosio,
Arturo
Falaschi, and
Alessandro
Vindigni
From the International Centre for Genetic Engineering and
Biotechnology, Padriciano 99, I-34012 Trieste, Italy
Received for publication, September 13, 2002, and in revised form, October 30, 2002
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ABSTRACT |
The RecQ helicases are involved in several
aspects of DNA metabolism. Five members of the RecQ family have been
found in humans, but only two of them have been carefully
characterized, BLM and WRN. In this work, we describe the enzymatic
characterization of RECQ1. The helicase has 3' to 5' polarity, cannot
start the unwinding from a blunt-ended terminus, and needs a
3'-single-stranded DNA tail longer than 10 nucleotides to open the
substrate. However, it was also able to unwind a blunt-ended duplex DNA
with a "bubble" of 25 nucleotides in the middle, as previously
observed for WRN and BLM. We show that only short DNA duplexes (<30
bp) can be unwound by RECQ1 alone, but the addition of human
replication protein A (hRPA) increases the processivity of the enzyme
(>100 bp). Our studies done with Escherichia coli
single-strand binding protein (SSB) indicate that the helicase activity
of RECQ1 is specifically stimulated by hRPA. This finding suggests that
RECQ1 and hRPA may interact also in vivo and function
together in DNA metabolism. Comparison of the present results with
previous studies on WRN and BLM provides novel insight into the role of
the N- and C-terminal domains of these helicases in determining their substrate specificity and in their interaction with hRPA.
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INTRODUCTION |
DNA and RNA helicases are a ubiquitous class of enzymes
characterized by their capacity to unwind and translocate along DNA or
RNA in reactions that are coupled to the binding and hydrolysis of a
5'-NTP (1). These enzymes are involved in most aspects of nucleic acid
metabolism, such as DNA replication, DNA repair, recombination,
transcription, RNA processing, and translation (2). Alterations of
genes that code for helicases cause several human disorders (3). For
example, two genes, XPB and XPD, encode for helicases that are
defective in individuals with xeroderma pigmentosum and Cockayne's
syndrome, respectively (4). Bloom's, Werner's, and Rothmund-Thomson
syndromes are additional genetic disorders that arise as a consequence
of abnormalities in three different members of the RecQ family of
helicases named BLM, WRN, and RECQ4, respectively (5-7). In all three
syndromes, cells from affected individuals show inherent genomic
instability, indicating that these RecQ helicases play a role in the
maintenance of chromosome stability.
The name RecQ derives from the first helicase of the family discovered
in Escherichia coli (8, 9). Successively, members of the
RecQ helicase family have been found in organisms that range from
bacteria to plants and animals (10, 11). In microorganisms like
E. coli, Saccharomyces cerevisiae, and
Schizosaccharomyces pombe, only one representative per
species is present, whereas higher eukaryotes contain more than one
RecQ helicase. For example, five members of the RecQ family have been
found so far in human cells, RECQ1, WRN, BLM, RECQ4, and RECQ5 (12).
All of them share a common central domain of ~450 amino acids
containing seven highly conserved motifs also present in several
helicases from other families (13). Among these motifs are an
ATP-binding sequence (Walker A box) and the DEXH box, which is
instead characteristic of the RecQ family. The RecQ helicases are
divided in two classes according to the length of the N- and C-terminal
domains. E. coli RecQ, human RECQ1, and RECQ5 form the
first group of RecQ helicases. They are characterized by short N- and
C-terminal domains, and their sequences are between 400 and 650 amino
acids long. The WRN, BLM, RECQ4, Sgs1p (from budding
yeast), and Rqh1p (from fission yeast) helicases are part of the second
group because they have extended N- and C-terminal tails and are all
between 1300 and 1500 amino acids long. The function of these extended
tails is still under investigation. One possibility is that the
additional portions mediate the interaction of these helicases with
other proteins. In fact, several proteins have been shown to interact with these longer helicases, such as replication protein A (14, 15),
proliferating cell nuclear antigen (16), DNA topoisomerase I (16), Ku
heterodimer (17-19), DNA polymerase
(20), and p53 (21, 22). A
common feature of all RecQ helicases studied so far is that they unwind
DNA with a 3' to 5' polarity. On the other hand, only the DNA-unwinding
activity and substrate specificity of the human BLM and WRN helicases
have been thoroughly investigated (22-26), whereas little or no
information is available so far on the catalytic properties of the
other three human helicases, RECQ1, RECQ4, and RECQ5 (27, 28).
In this work, we describe the enzymatic characterization of the
helicase activity of human RECQ1. The protein was purified from HeLa
cells following a procedure similar to the one established in our
laboratory for the purification of several other human DNA helicases
(29-32). The possibility that the helicase activity of RECQ1 may be
specifically stimulated by human replication protein A
(hRPA),1 as in the case of
the Werner's and Bloom's helicases, was also investigated. Comparison
of our results with the previously reported hRPA stimulation of the WRN
and BLM helicases provides novel insight into the roles that the
N- and C-terminal tails and the central 450-amino acid domain of these
RecQ helicases play in the interaction with hRPA.
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EXPERIMENTAL PROCEDURES |
Reagents--
All salts, bovine serum albumin, dithiothreitol,
phenylmethylsulfonyl fluoride, leupeptin, and pepstatin were from
Sigma. The M13mp18 single-stranded DNA (ssDNA) plasmid, the serum used to grow the HeLa cells, glutamine, and gentamycin were from Invitrogen. All resins used for the different purification steps were from Amersham
Biosciences (Uppsala, Sweden). Most of the purification steps were
carried out using an AKTA FPLC system (Amersham Biosciences). Gel filtration studies were performed with a TSK-GEL G3000SW
column (60 cm × 7.5 mm; TOSOH BIOSEP, Stuttgart, Germany).
All ssDNA oligonucleotides used to make the DNA substrates were
purchased from Sigma (Cambs, UK). The radioactive nucleoside
triphosphates were obtained from Amersham Biosciences (Buckinghamshire,
UK). The T4 polynucleotide kinase and sequencing grade porcine trypsin for protein digestion were from Promega (Madison, WI). Recombinant hRPA
containing all three subunits (RPA70, RPA32, and RPA14) was expressed
in and purified from E. coli according to the previously described protocol (33).
Cell Culture and Buffers--
HeLa cells were grown in
Joklin minimal essential medium supplemented with 10%
fetal calf serum, 50 µg/ml gentamycin, and 2 mM glutamine
and harvested as described previously (31). All buffers used during the
purification of RECQ1 contained 1 mM dithiothreitol and 0.5 mM phenylmethylsulfonyl fluoride as protease
inhibitor. Buffer A contained 20 mM HEPES (pH 8.0), 0.1 M NaCl, 1 mM EDTA, and 20% glycerol. Buffer B
contained 50 mM Tris-HCl (pH 7.5), 70 mM KCl, 1 mM EDTA, and 10% glycerol. The concentration of NaCl or
KCl in all buffers was increased up to 1.0 M for eluting
the proteins from the different columns.
Purification of RECQ1--
The RECQ1 helicase
was purified from 300 g of frozen HeLa cells. The cell nuclei were
isolated and salt-extracted with 0.4 M NaCl following the
procedure described by Dignam et al. (34). Successively, an
additional salt extraction with 1.0 M NaCl was done to
select specifically for proteins that bind tightly to DNA. The
extracted proteins were precipitated by slowly adding ammonium sulfate
(0.35 g/ml), collected by centrifugation at 25,000 × g
for 30 min in a Sorvall SS34 rotor, dialyzed in buffer A, and applied
to a Bio-Rex column (2.5 × 33 cm) equilibrated with buffer A
(35). Active fractions eluting at ~0.4 M NaCl in buffer A
were pooled (Fraction I, 78 ml). All purification steps were carried
out at 4 °C, and the unwinding activity after each step of
purification was monitored with a 5'-32P-labeled DNA
substrate as described (29). Fraction I was first dialyzed in buffer B
and then loaded onto a 10-ml Q-Sepharose column equilibrated with
buffer B. The proteins bound to the column were eluted using a linear
gradient of 0.07-1.0 M KCl in buffer B. All active
fractions eluting at the very beginning of the gradient were pooled
(Fraction II, 85 ml) and loaded onto a 1-ml Mono S column. Elution was
carried out with the same linear gradient used for the Q-Sepharose
column, and the helicase eluted between 0.2 and 0.3 M KCl
(Fraction III, 14 ml). Fraction III was dialyzed in buffer B and loaded
onto a 4-ml ssDNA-cellulose column (1.6 × 2.5 cm) for the last
step of purification. Elution was carried out with a linear gradient of
5 column volumes of 0.07-1.0 M KCl in buffer B. A major
peak eluted at ~0.24 M KCl. The active fractions were
pooled together (Fraction IV, 8 ml, 44.800 units) and stored at
80 °C with 50% glycerol.
Preparation of DNA Helicase Substrates--
The DNA substrates
used in the helicase assay are listed in Fig. 5. They consist of
different 32P-labeled oligonucleotides annealed either to
M13mp18 phage ssDNA or to ssDNA fragments of different lengths to
create a partial duplex. The sequence of the 99-bp oligonucleotide used
for the determination of the direction of unwinding (see Fig. 5,
A and B) is the same as that described previously
(36), as are the sequences of three oligonucleotides annealed to the
M13mp18 phage ssDNA (see Fig. 5, D-G) (29, 36). The
substrate in Fig. 5H was made using oligonucleotide
5'-CTCTAGAGGATCCCCGGGTACCGAGCTCGAATT-3' (33 bp), complementary to
nucleotides (nt) 6231-6263 of M13mp18 phage ssDNA. The blunt-ended
25-bp duplex (see Fig. 5C) was made by annealing
oligonucleotide 5'-GATCTCGCATCACGTGACGAAGATC-3' to its complement. The
substrate in Fig. 5I was made by annealing oligonucleotide
5'-GATCTCGCATCACGTGACGATTTTTTTTTTTTTTTTTTTTTTTTTGATCTCGCATCACGTGACGA-3' to a ssDNA fragment complementary to the oligonucleotide except for the
25 T stretch in the middle. Linear substrates with poly(dT) tails of different lengths were made using a 32P-labeled
29-bp oligonucleotide (5'-GTCAAATAGCAAACATGAAAGCATAAAAC-3') annealed to complementary ssDNA fragments with 3'-tails of 10, 25, 50, and 75 dT nucleotides (see Fig. 5, K-N). The substrate with
a double-strand region of 109 bp was made by PCR amplification of a
M13mp18 fragment of the proper length. The forward primer for the PCR
was annealed to region 28-47 of M13mp18, whereas the reverse primer
was annealed to region 114-137. PCR conditions were as follows: 10 mM Tris-HCl, 50 mM KCl, 0,1% Triton X-100, 2 mM MgCl2, 200 µM dNTPs, 1 µM each primer, and 2.5 units of Taq polymerase (Promega) in a 50-µl reaction. Cycling conditions were as
follows: initial denaturation at 95 °C for 2 min; 35 cycles at
95 °C for 30 s, 65 °C for 30 s, and 72 °C for
30 s; and finally, elongation at 72 °C for 7 min. For all
substrates, 25 ng of each oligonucleotide, labeled at the 5'-end with
T4 polynucleotide kinase and 0.9 MBq of [
-32P]ATP,
were subsequently annealed to M13mp18 phage ssDNA (4 µg) in 40 mM Tris-HCl (pH 8.0), 10 mM MgCl2,
1 mM dithiothreitol, and 50 mM NaCl. The
mixture was heated at 95 °C for 2 min and slowly cooled to room
temperature. Each substrate was purified by gel filtration through a
5-ml Sepharose 4B column.
Preparation of RNA/RNA Substrate--
The RNA/RNA
substrate was obtained as follows. The pDEST17 vector (Invitrogen)
containing the annexin II gene was linearized by EcoRV and
transcribed in vitro with T7 RNA polymerase (Promega) from
the specific promoter, yielding a 2.2-kb RNA (30). A 16-bp RNA
oligonucleotide complementary to the same region of annexin II (nt
53-68) was synthesized and labeled at the 5'-end with T4 polynucleotide kinase and 9.25 MBq of [
-32P]ATP. About
8 pmol of labeled oligonucleotide were mixed with 13 pmol of
synthesized annexin II RNA, heated at 95 °C for 1 min, and allowed
to anneal by slow cooling. The substrates were then purified by gel
filtration through a 5-ml Sepharose 4B column.
DNA Helicase Assay--
The helicase assay measures the
unwinding of a
-32P-labeled DNA fragment from a partial
duplex DNA molecule. The 10-µl reaction mixture contained 20 mM Tris-HCl (pH 9.0), 8 mM dithiothreitol, 2 mM MgCl2, 3 mM ATP, 10 mM KCl, 4% (w/v) sucrose, 80 µg/ml bovine serum albumin
(BSA), and 32P-labeled helicase substrate (1000 cpm, 1 ng,
~0.05 nM). The helicase fraction to be assayed was added
to the mixture and incubated at 37 °C for 30 min, and the reaction
was terminated by the addition of 0.3% SDS, 10 mM EDTA,
5% glycerol, and 0.1% bromphenol blue. The products of the reaction
were fractionated by electrophoresis on a 12% nondenaturing
polyacrylamide gel. The gel was dried, and the extent of DNA unwinding
was quantitated by electronic autoradiography (Instant Imager, Packard
Instrument Co.). One unit of DNA helicase is defined as the amount of
enzyme unwinding 1% of the substrate in 1 min at 37 °C (30% in 30 min) in the linear range of enzyme concentration dependence.
Native Molecular Mass Determination--
The native molecular
mass was determined by glycerol gradient centrifugation and gel
filtration analysis following the procedure described previously (29,
37, 38). More precisely, for the glycerol gradient study, 100 µl of
50 nM RECQ1 were layered on a 15-35% glycerol gradient in
buffer B and centrifuged at 320,000 × g for 20 h
at 4 °C. The standard protein markers were also run under the same
conditions. The markers were catalase (240 kDa, 11.3 S), aldolase (158 kDa, 7.4 S), BSA (66 kDa, 4.22 S), and ovalbumin (45 kDa, 3.5 S).
Fractions of 0.2 ml were collected from the top of the tube using an
HSI Auto Densi-Flow IIC apparatus (Buchler Instruments) and assayed for
helicase activity. For gel filtration, a TSK-GEL G3000SW column (60 cm × 7.5 mm) was used in the AKTA FPLC system equilibrated with
buffer B. The solution of RECQ1 was first concentrated from 50 to 500 nM and then loaded onto the column. The column was run at a
flow rate of 1 ml/min. Fractions of 0.25 ml were collected and assayed
for helicase activity. The column was pre-calibrated using gel
filtration molecular mass markers under the same conditions. The
markers were thyroglobulin (670 kDa, Stokes radius of 85 Å),
-globulin (158 kDa, 48.1 Å), BSA (66 kDa, 35.5 Å), ovalbumin (45 kDa, 30.5 Å), myoglobin (17 kDa, 21.2 Å), and vitamin B12
(1.35 kDa). The partition coefficient Kav is
equal to (Ve
V0)/(Vt
V0), where Ve is the elution
volume of the sample, V0 is the void volume, and Vt is the total volume of the gel bed. The Stokes
radius of RECQ1 was derived from the linear plot of (
log
Kav)1/2 versus the Stokes
radius of the standard proteins. The molecular mass of RECQ1 was
calculated from the Stokes radius and the sedimentation coefficient
using the equation previously described (38).
Microsequence Analysis--
The Coomassie Blue- and
silver-stained bands containing RECQ1 were cut out and digested
with bovine trypsin (Promega). The digestion products were separated by
micro-high pressure liquid chromatography and analyzed by
electrospray ionization mass spectrometry (Finnigan LCQ DECA,
Thermo-Finningan Corp., San Jose, CA).
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RESULTS |
The human helicase RECQ1 was purified from the nuclear extract of
HeLa cells following the purification steps described under "Experimental Procedures." The final product was loaded on a 10% SDS-acrylamide gel for analysis of its purity. The silver-stained gel
showed only a single band of ~70 kDa (Fig.
1). Successively, the band was excised
from the gel, digested with trypsin, and analyzed by mass spectrometry
for protein identification. Ten peptides pertaining to the human DNA
helicase RECQ1 (75 kDa) were found in the sample (Table
I). The five helicases of the RecQ family
that have been found in human cells are characterized by a conserved
central domain of ~450 amino acids. On the other hand, the 10 peptides found by mass spectrometry have sequences that are unique for
RECQ1, allowing us to be certain about the identity of the protein. Six
peptides correspond to sequences located in the central domain of the
protein, two in the N-terminal domain, and two in the C-terminal tail
(Fig. 2).

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Fig. 1.
SDS-polyacrylamide gel of purified RECQ1.
Lane 1, molecular mass markers (in daltons); lane
2, purified RECQ1 (100 ng). The 10% acrylamide gel was stained
with silver for better detection of eventual impurities. No additional
bands were detected after loading 2 µg of RECQ1 on the gel.
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Fig. 2.
RecQ helicase family. Shown is a
schematic representation of some members of the RecQ family of
helicases from E. coli to human. The conserved central
domain in each helicase is shown as a black box. The
white boxes indicate the positions of the peptides of RECQ1
found by mass spectrometry.
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After its identification, we determined the native molecular mass
of RECQ1 by glycerol gradient and gel filtration following a
previously described procedure (38, 39). The result indicates that
RECQ1 has a sedimentation coefficient of 7.3 ± 1.7 S and a Stokes
radius of 49.5 ± 10.5 Å, corresponding to a native molecular mass of 160 ± 18 kDa, thus suggesting that the protein exists as
a dimer in solution (Fig. 3). On the
other hand, further studies will need to be done under different buffer
conditions and with additional techniques to obtain more accurate
information on the oligomerization state of this molecule.
Concentration dependence studies under optimal assay conditions showed
a maximum value of ~100% unwinding in 30 min with 4 nM
enzyme (Fig. 4A). The
sigmoidal shape at the very beginning of the titration curve is
indicative of cooperative behavior, suggesting that more than one
molecule of RECQ1 could be involved in DNA unwinding, as seen in the
case of other helicases (1). Kinetic measurements carried out in the
presence of 1 nM enzyme (740 pg) showed that the unwinding rate was linear for up to 10 min and deviated from linearity with longer incubation times (Fig. 4B). The helicase
assays indicated that ATP and Mg2+ are indispensable
for DNA unwinding. In addition, ATP dependence studies
indicated that the optimal ATP concentration for DNA unwinding is
between 4 and 5 mM (data not shown). Interestingly, we also observed that the addition of 80 µg/ml BSA increased the unwinding activity of RECQ1. This observation could be explained by previous studies showing that the presence of BSA increases the affinity of some
proteins for DNA (40, 41).

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Fig. 3.
Glycerol gradient (15-35%) sedimentation
and gel filtration analysis. A, the experiment was
performed using 100 µl of 50 nM RECQ1, and centrifugation
was performed at 320,000 × g for 20 h at 4 °C.
The distribution of the helicase activity, the positions of the
sedimentation coefficients, and molecular mass markers are shown. The
markers were catalase (240 kDa, 11.3 S), aldolase (158 kDa, 7.4 S), BSA
(66 kDa, 4.22 S), and ovalbumin (45 kDa, 3.5 S). B, gel
filtration was carried out using of 100 µl of RECQ1 (500 nM) on a TSK-GEL G300SW column (60 cm × 7.5 mm). The
distribution of the helicase activity and the positions of the
molecular mass markers are shown. The markers were thyroglobulin (670 kDa), -globulin (158 kDa), BSA (66 kDa), ovalbumin (44 kDa),
myoglobin (17 kDa), and vitamin B12 (1.35 kDa).
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Fig. 4.
Concentration and time dependence of RECQ1
activity. A, increasing amounts of RECQ1 were incubated
in the standard 10-µl reaction mixture for 30 min at 37 °C. The
concentration of enzyme (nanomolar) is indicated above each lane in the
autoradiogram, whereas the substrate concentration was constant in all
experiments (0.4 nM). B, the kinetics of
unwinding were performed using 1 nM RECQ1 and the same
reaction conditions used for the concentration dependence experiments.
The reaction times (in minutes) are indicated above each lane in the
autoradiogram. The C and D lanes are
control assays without enzyme and heat-denatured substrate,
respectively.
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The only information that was available on the helicase activity of
human RECQ1 is that it unwinds DNA with 3' to 5' polarity, like the
other members of the RecQ family characterized so far (42). This
polarity of unwinding was confirmed by our experiments showing that
RECQ1 needs a 3'-ssDNA tail to unwind the substrate (Fig.
5, A-C). Following this
observation, a set of substrates with different structures and with
various lengths of the double-strand regions were prepared to obtain
novel information on the substrate specificity of RECQ1 (Fig.
5). Substrates with a 17-bp oligonucleotide annealed to M13 ssDNA were
fully unwound by RECQ1, regardless of the presence or absence of
mismatched hanging tails at the 5'-end, the 3'-end, or both (Fig. 5,
D-G). However, if the duplex region was increased to 33 bp,
the substrate could not be unwound (Fig. 5H). Similarly to
what has been already observed for the BLM and WRN helicases (43),
RECQ1 was able to unwind a substrate with a "bubble" of 25 nt
located in the center (Fig. 5I). Other helicases previously
purified in our laboratory have been shown to be able to unwind DNA/DNA
as well as RNA/RNA duplexes (29); in contrast, RECQ1 could
not unwind RNA substrates (Fig. 5J), indicating that it can
work only as a DNA helicase. Finally, a series of linear substrates
with 3'-single-strand tails of 10, 25, 50, and 75 dT were made to study
the effect of tail length on the unwinding activity of RECQ1 (Fig. 5,
K-N). Kinetic studies done with this series of
substrates clearly showed that only the substrates with tails longer
than 10 nt could be efficiently unwound by RECQ1 (Fig.
6).

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Fig. 5.
Helicase activity with various
substrates. Each panel shows the structure of the substrate used
and an autoradiogram of the gel. Asterisks denote the
32P-labeled end. Percent unwinding is shown above each
autoradiogram. Lanes 1 and 3 correspond to
control reactions without enzyme and heat-denatured substrate,
respectively. Lane 2 corresponds to the reaction with pure
RECQ1 (~1.0 nM). The direction of unwinding is analyzed
in A and B.
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Fig. 6.
Tail length dependence studies. The
kinetics of unwinding were determined with three DNA substrates having
poly(T) tails of 10 ( ), 25 ( ), and 75 ( ) nt. The concentration
of RECQ1 used in all kinetic studies was 1 nM. The reaction
times (in minutes) are indicated above each lane in the autoradiograms.
The C and D lanes are control assays
without enzyme and heat-denatured substrate, respectively.
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hRPA specifically stimulates the DNA-unwinding activity of the WRN and
BLM helicases (14, 15). In our experiments, we saw that also the
helicase activity of RECQ1 was specifically stimulated by the addition
of hRPA. The RECQ1 helicase (1 nM) alone could not unwind a
DNA substrate with a duplex region of 33 bp, but with the addition of
hRPA or E. coli single-strand binding protein (ESSB), the
substrate was unwound. Kinetic and concentration dependence studies
showed that, in the presence of 0.3 µM hRPA, the
unwinding reaction was complete in <15 min (Fig.
7, A and B). On the
other hand, even at a concentration of ESSB (3 µM)
10-fold higher than that of hRPA (0.3 µM), only 40% of
the substrate was unwound after 1 h. To gain more insight into the
mechanism of stimulation by these single-strand binding proteins,
strand displacement was expressed as a function of the ratio
(R) of the concentration of single-strand binding protein units to the concentration of DNA-binding units (given by the concentration of the ssDNA substrate in nucleotides divided by the
number of oligonucleotides covered by each unit). In this way, the new
analysis takes into account that hRPA covers ~30 nt when binding to
DNA (44), whereas ESSB binds ~35 nt (45) (Fig. 7C). The
plot shows that, at a concentration of hRPA that coated the ssDNA
molecules in the helicase reaction (96 nM heterotrimer), 15% of the 33-mer was unwound, whereas no unwinding was detectable at
a coating concentration of ESSB (82 nM). Similarly, at an
R value of 3, 100% of the duplex was released in the
presence of hRPA, but no unwinding was observed with ESSB, suggesting
that, even at high R values, ESSB poorly stimulates RECQ1
helicase activity. This observation was further supported by the
results obtained with the 109-bp duplex (Fig.
8). This long substrate could be unwound
only when hRPA was present, whereas ESSB failed to catalyze the
unwinding even at a concentration 10-fold higher than that used for
hRPA and after 3 h of reaction.

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Fig. 7.
Stimulation of the helicase activity of RECQ1
by hRPA and ESSB. The substrate used in all experiments was the
33-bp partial duplex shown in Fig. 5H, and the concentration
of RECQ1 was always 1 nM. A, the kinetics of
unwinding were determined using RECQ1 alone ( ) and in the presence
of 0.3 µM hRPA ( ) or 3 µM ESSB ( ).
The reaction times (in minutes) are indicated above each lane in the
autoradiograms. B, concentration dependence studies were
carried out at increasing concentrations of hRPA ( ) and ESSB ( ).
The protein concentrations (nanomolar) are indicated above each lane in
the autoradiograms. C, the percentage of unwinding is
expressed as a function of R, defined as the ratio of the
concentration of single-strand binding protein units to the
concentration of DNA-binding units.
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Fig. 8.
Kinetics of unwinding of a 109-bp partial
duplex substrate in the presence of hRPA. The
kinetics of unwinding were determined using RECQ1 alone ( )
and in the presence of 0.3 µM hRPA ( ) or 3 µM ESSB ( ). The concentration of RECQ1 was always 1 nM. The reaction times (in minutes) are indicated above
each lane in the autoradiograms.
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DISCUSSION |
The physiological role of the five members of the RecQ
family of helicases found in humans is still uncertain and under
debate (12, 46). Several reports indicate that the human
RecQ helicases play a key role in the maintenance of chromosome
stability and point to their possible roles in different aspects of DNA
metabolism, such as DNA replication, double-strand break repair,
telomere maintenance, and homologous recombination (12, 46). In any case, deletion of the only RecQ family gene in S. cerevisiae
leads to a significant impairment of DNA metabolism, but does not cause cell death (47). This also applies to the human cell mutants (48); but,
in this case, it is conceivable that other members of the family
partially rescue for possible essential function. Information
on the substrate specificity of RecQ helicases and on their mechanism
of DNA unwinding is essential for a better understanding of their
function. On the other hand, only the enzymatic activity of two human
RecQ helicases, WRN and BLM, has been carefully investigated to date
(48). We isolated the human RECQ1 helicase from HeLa nuclear extracts
and identified it by mass spectrometry; this is one of the first
helicases of the RecQ family discovered in human cells, and very little
information on its catalytic and molecular properties was available
(49, 50). The only information available on the oligomerization state
of the RecQ helicases comes from studies performed on the BLM protein,
where it was shown, by electron microscopy and size-exclusion
chromatography, that it forms hexameric structures in solution (51). On
the other hand, our observation that RECQ1 seems to form dimers rather
than hexamers suggests that the five helicases of the RecQ family may adopt different structures and may follow diverse mechanisms to unwind
DNA. In addition, the sigmoidal shape of the titration curve as a
function of increasing RECQ1 concentrations is indicative of
cooperative behavior, suggesting that more than one molecule of RECQ1
could be involved in DNA unwinding, as seen in the case of other
helicases (1); thus, the possibility that a hexameric structure builds
up on the appropriate substrate cannot be ruled out.
The 3' to 5' polarity of unwinding was the only information available
on the helicase activity of RECQ1 (42) and has been also confirmed by
our results. The substrate specificity of RECQ1 was investigated with a
series of DNA probes of different structures and lengths. As was
previously observed for the BLM and WRN helicases (43), RECQ1 cannot
unwind blunt-ended DNA substrates. In contrast, the E. coli
RecQ helicase is able to initiate duplex unwinding from a
blunt-ended terminus (52). Nevertheless, RECQ1 is able to unwind
certain kinds of blunt-ended duplexes because it could unwind a
blunt-ended DNA substrate with a 25-bp bubble in the center
(Fig. 5), again in agreement with what had been previously observed for
WRN and BLM (43). Based on the short size of its N- and
C-terminal domains, RECQ1 is the human RecQ helicase, among the three
(BLM, WRN, and RECQ1), that most resembles the E. coli variant. For this reason, it could have been predicted that RECQ1 would
have greater similarity to E. coli RecQ than the other two human helicases. On the other hand, our findings suggest that the
residues responsible for the substrate specificity of these enzymes
must reside in the conserved central domain rather than in the N- and
C-terminal domains and indicate that RecQ helicases from different
organisms have different substrate specificities. We also studied the
effect of the length of the 3'-ssDNA tails on the unwinding activity of
RECQ1 (Fig. 5). Tail length studies have not been performed
before on other human RecQ helicases, but it was shown that
Sgs1p from S. cerevisiae is able to unwind substrates with
3'-tails of only 3 nt. Our results indicate that RECQ1 differs from
Sgs1p because a DNA probe with a 3'-tail of 10 nt was poorly unwound
and only when the tail length was increased to 25 nt >70% of the
substrate was unwound (Fig. 5). A possible explanation is that RECQ1
needs a ssDNA tail longer than 10 nt to efficiently bind the substrate
and to start the unwinding.
A common feature of the human RecQ helicases is that they
are not able to unwind long DNA duplexes, but recent reports have shown
that the addition of hRPA significantly enhances the processivity of
WRN and BLM (14, 15). In line with these results, we observed that
RECQ1 alone was unable to unwind a 33-bp duplex DNA probe under our
experimental conditions, but the substrate could be easily unwound if
hRPA was added to the reaction mixture. Upon addition of ESSB, the
substrate could also be unwound, although much less efficiently
because, even using 10-fold more ESSB than hRPA, only 40% of the
duplex was unwound. The greater ability of hRPA over ESSB in
stimulating the helicase activity of RECQ1 suggests that hRPA performs
an additional role in the unwinding reaction rather than simply coating
the single strands generated during the opening of the duplex. This
conclusion is strengthen by the results obtained with the 109-bp
partial duplex substrate. Helicase assays done in the presence of hRPA
or ESSB showed that hRPA was absolutely required for the unwinding of
the 109-bp duplex region, whereas ESSB was completely unable to
stimulate the RECQ1-catalyzed unwinding reaction of this long
substrate. These data indicate that both proteins, hRPA and RECQ1, are
necessary for the unwinding of long substrates and suggest that these
two proteins may functionally interact in vivo. A specific
interaction between RECQ1 and hRPA could indicate a potential role of
this helicase in replication, recombination, or repair, all processes
in which hRPA has been shown to be involved (44).
Our findings also provide novel information on the roles of
the N- terminal, C-terminal, and central domains of the RecQ helicases in the interaction with hRPA. Previous studies have shown that the N
terminus of WRN contains a 3'
5' exonuclease domain and mediates
the interaction of WRN with the Ku70 subunit (17, 19) and proliferating
cell nuclear antigen (16), whereas the C terminus is responsible for
the interaction with p53 (21, 53) and the Ku80 subunit (19). The
extended N- and C-terminal domains of the BLM helicase mediate its
interaction with topoisomerase III (54) and RAD51 (55). On the other
hand, no information on the roles of the N- and C-terminal domains of
these helicases in the interaction with hRPA is available. The
observation that both WRN and BLM physically interact with hRPA could
lead to the conclusion that the extended N- and C-terminal domains of
these two helicases may also be involved in this binding event.
However, our data suggest that the central domain of these RecQ
helicases must be the one involved in the interaction of RECQ1, WRN,
and BLM with hRPA because RECQ1 lacks the extended N- and C-terminal domains of WRN and BLM.
The picture that emerges from our studies is that the substrate
specificity and DNA-unwinding activity of RECQ1 are, in many aspects,
similar to those of the BLM and WRN helicases, although different from
those of E. coli RecQ and budding yeast Sgs1p. Also the
discovery that RECQ1 interacts with hRPA is in agreement with previous
studies done with BLM and WRN. These similarities allow us to conclude
that the extended N- and C-terminal domains of BLM and WRN are not
responsible for the substrate specificity of these helicases and are
not involved in the interaction of these proteins with hRPA. The fact
that all three helicases interact with hRPA suggests that they could be
involved in the same physiological processes and work in a
complementary fashion, so the absence of one of them could be partially
compensated for by the presence of the other. This hypothesis is also
supported by other studies showing that WRN and BLM physically interact
(23) and are both involved in DNA repair in a complementary fashion
(56). On the other hand, the observation that WRN and BLM, with their
extended N- and C-terminal domains, can also interact with other
partners, such as p53, Ku, and topoisomerases, indicates that they can
also be involved in some specific physiological functions where RECQ1 is not required. However, more studies will need to be done to reach a
better understanding of the physiological roles of this intriguing
family of human helicases.
 |
ACKNOWLEDGEMENTS |
We are indebted to Dr. Mark Wold for
providing purified hRPA and the plasmid for the expression of hRPA in
E. coli. The assistance of Maria Elena Lopez and Oscar
Sandoval in the preparation of HeLa cells is gratefully acknowledged.
We thank Marlen Lujardo Gonzàlez for assistance in the mass
spectrometry analysis.
 |
FOOTNOTES |
*
The work was supported by Grant 99.00649.PF33 from the
Consiglio Nazionale delle Ricerche, Roma.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 39-040-375-7326;
Fax: 39-040-226-555; E-mail: vindigni@icgeb.org.
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M209407200
 |
ABBREVIATIONS |
The abbreviations used are:
hRPA, human
replication protein A;
ssDNA, single-stranded DNA;
nt, nucleotides;
BSA, bovine serum albumin;
ESSB, E. coli single-strand
binding protein.
 |
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