Biochemical analysis of the DNA unwinding and strand annealing activities catalyzed by human

35 Pages·2005·2.65 MB·English

JBC Papers in Press. Published on May 16, 2005 as Manuscript M500264200
Biochemical Analysis of the DNA Unwinding and Strand Annealing
Activities Catalyzed by Human RECQ1

Sudha Sharma1, Joshua A. Sommers1, Saba Choudhary1, Jinnifer Korin Faulkner1,
Sheng Cui2, Lucia Andreoli2, Laura Muzzolini2,
Alessandro Vindigni2, and Robert M. Brosh, Jr.1*
From the 1Laboratory of Molecular Gerontology, National Institute on Aging, NIH, 5600
Nathan Shock Drive, Baltimore, MD 21224, USA, 2International Centre for Genetic
Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy
Running Title: Biochemical Analysis of Human RECQ1
*Please send correspondence to: Robert M. Brosh, Jr., Laboratory of Molecular
Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore,
Maryland 21224 USA, Phone: [phone], FAX: [phone] E-mail:
[email protected]
RecQ helicases play an important ATP binding, which induces a
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role in preserving genomic integrity, and conformational change in the protein. The ow
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their cellular roles in DNA repair, enzymatic properties of RECQ1 helicase lo
a
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recombination, and replication have been of and strand annealing activities are e
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considerable interest. Of the five human discussed in the context of proposed cellular fro
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RecQ helicases identified, three are DNA metabolic pathways that are h
associated with genetic disorders important in the maintenance of genomic ttp://w
characterized by an elevated incidence of stability. w
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cancer or premature aging: Werner .jb
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syndrome (WS), Bloom syndrome (BS), and Cellular processes such as DNA .o
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Rothmund-Thomson Syndrome (RTS). replication, recombination, and repair often b/
y
Although the biochemical properties and involve steps that require unwinding of g
u
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protein interactions of the WRN and BLM double-stranded (ds) DNA to form transient st o
helicases defective in WS and BS, single-stranded (ss) DNA intermediates. n N
respectively, have been extensively Helicases are a class of enzymes that unwind ov
e
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investigated, less information is available DNA duplexes with a distinct directional b
e
concerning the functions of the other polarity, either 3’ to 5’ or 5’ to 3’ with respect r 1
7
human RecQ helicases. We have focused to the strand on which the helicase is , 2
0
our attention on human RECQ1, a DNA presumed to translocate, deriving energy from 18
helicase whose cellular functions remain the hydrolysis of ATP (1;2). An essential
largely uncharacterized. In this work, we “caretaker” role of helicases in DNA
have characterized the DNA substrate metabolic processes is suggested by a number
specificity and optimal cofactor of genetic disorders that have been linked to
requirements for efficient RECQ1 defects in DNA helicases (3-5).
catalyzed DNA unwinding, and determined The RecQ family of DNA helicases,
that RECQ1 has certain properties that are named after the Escherichia coli RecQ DNA
distinct from other RecQ helicases. RECQ1 helicase, is highly conserved from bacteria to
stably binds to a variety of DNA structures, humans (4). RecQ helicases share a centrally
enabling it to unwind a diverse set of DNA located helicase domain of ~450 residues
substrates. In addition to its DNA binding containing the seven conserved helicase motifs
and helicase activities, RECQ1 catalyzes and an additional conserved region of ~80
efficient strand annealing between amino acids located C-terminal to the helicase
complementary single-stranded DNA domain designated RecQ-Ct (RQC) (4;6). The
molecules. The ability of RECQ1 to conserved helicase domain couples nucleotide
promote strand annealing is modulated by hydrolysis to DNA unwinding, while the RQC
1
domain is suggested to be involved in protein steps. Although the precise details of BLM
interaction (7) and DNA binding (8). In all function in SDSA are not well understood, the
organisms, RecQ helicases play critical roles in ability of BLM to unwind D-loop structures
maintaining genomic stability as evidenced by (13), a key intermediate of the SDSA pathway,
the hyper-recombination phenotype observed in is likely to be relevant. Consistent with this,
recQ mutants (for review, see (9)). the Drosophila genetic data support a model in
There are five human helicases with which BLM acts downstream of strand
the defined region of homology in the invasion (12). Understanding the molecular
conserved RecQ helicase motifs: 1) WRN, functions of RECQ1 might provide some
defective in Werner syndrome (WS); 2) BLM, insight to how the enzyme has at least partially
defective in BLM syndrome (BS); 3) RTS, redundant or synergistic functions with BLM
defective in Rothmund-Thomson syndrome in a pathway such as SDSA.
(RTS); 4) RECQ1; and 5) RECQ5 (3). WS, Recent biophysical and biochemical
BS, and RTS are all characterized as genomic characterization of the purified recombinant
instability disorders with an elevated cancer RECQ1 helicase provides evidence that
incidence and/or premature aging. Human RECQ1 forms dimers in solution (14) and is
diseases have not yet been genetically linked able to unwind short 3’ ssDNA tailed duplex
D
to mutations in RECQ1 or RECQ5; however, DNA substrates (14;15). Considerably longer o
w
n
these proteins are likely to have important substrates were shown to be unwound in the lo
a
roles in nucleic acid metabolism based on the presence of the human single-stranded DNA de
d
biological significance of other RecQ binding protein Replication Protein A (RPA) fro
m
helicases. (14;15). In the current study, we have h
The biological importance of RECQ1 investigated the unwinding of various DNA ttp://w
cannot be clearly evaluated at present since structures by RECQ1 helicase. In the course w
w
there is little information on the phenotypes of of this work, we determined that RECQ1 has .jb
human RECQ1 mutant cells. Recent genetic the ability to form stable protein complexes c.o
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complementation studies using the chick with either ssDNA or dsDNA, suggesting that b/
y
DT40 cell-based system indicate that RECQ1 RECQ1 may utilize its physical interaction g
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(-/-) cells do not display a growth deficiency, with DNA for functions other than duplex es
t o
sensitivity to DNA damage, or elevated sister DNA unwinding. Recently, a novel strand n
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chromatid exchange; however, RECQ1(-/-) annealing activity of human RECQ5β helicase o
v
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BLM(-/-) cells grow more slowly than BLM (- was reported (16). Since RECQ1 was able to m
b
e
/-) cells due to an increased population of dead efficiently bind ssDNA, we became interested r 1
7
cells, indicating that RECQ1 is involved in in the possibility that RECQ1 might utilize its , 2
0
cell viability under the BLM-impaired ssDNA binding activity to bridge the 18
condition (10). In addition, the double mutant complementary ssDNA molecules and
RECQ1(-/-) BLM(-/-) cells exhibit elevated promote strand annealing. The results
mitomycin C-induced sister chromatid presented here provide evidence that RECQ1
exchange compared to the BLM(-/-) cells, can efficiently catalyze DNA unwinding of
suggesting that RECQ1 may have some model recombination/repair DNA
redundant functions with the BLM helicase intermediates or promote strand annealing of
(10). complementary ssDNA molecules, and that
Recent studies of BLM function in these processes are modulated by nucleotide
Drosophila suggests a role of the helicase in availability which alters RECQ1 protein
double strand break repair by a homologous conformation. The dual catalytic functions of
recombination pathway known as synthesis- RECQ1 are likely to be important in a cellular
dependent strand annealing (SDSA) (11;12). DNA metabolic pathway important for
This pathway exploits the cellular maintenance of genomic stability.
recombination machinery to rejoin double
strand breaks through a series of strand
invasion, DNA synthesis, and reannealing
2
MATERIALS AND METHODS unlabeled competitor oligonucleotide to
prevent reannealing of the unwound single-
Proteins-Recombinant His-tagged RECQ1 stranded DNA products. Unlabeled
protein was over-expressed using a competitor oligonucleotide was not included
baculovirus/Sf9 insect system and purified to in the quench for helicase reactions with D-
near homogeneity as previously described loop or HJ substrates. Reaction products were
(14). A baculovirus encoding recombinant subsequently incubated with 0.1 mg/ml
His-tagged RECQ-K119A mutant protein was Proteinase K for 15 min at 37oC, and the
constructed as described elsewhere (17), and products of standard duplex and D-loop
the RECQ1-K119A protein was purified using substrates were resolved on nondenaturing
the same procedure as for the wild-type 12% polyacrylamide gels. Products of the HJ
RECQ1 recombinant protein. Purified branch migration reactions were resolved on
recombinant human RPA was kindly provided native 10% polyacrylamide gels.
by Dr. Mark Kenny (Albert Einstein Medical Radiolabeled DNA species in polyacrylamide
Center, Bronx, NY). Purified recombinant gels were visualized using a PhosphorImager
RuvA protein was kindly provided by Dr. and quantitated using ImageQuant software
Michael Cox (University of Wisconsin, (Amersham Biosciences). The percent
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Madison, WI). E. coli Single-stranded DNA helicase substrate unwound was calculated by o
w
Binding Protein (ESSB) was purchased from the formula: percent unwinding = 100 x (P/(S nlo
a
Promega. + P)) where P is the product and S is the de
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DNA Substrates- PAGE-purified residual substrate. The values of P and S have fro
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oligonucleotides used for preparation of DNA been corrected after subtracting background h
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DNA substrates were prepared as described data represent the mean of at least three w.jb
previously (18). Synthetic Holliday Junction independent experiments with mean ± standard c.o
HJ(X12) was made by annealing four 50-mer deviation (SD) indicated by error bars. brg/
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oligonucleotides (X12-1, X12-2, X12-3, X12- DNA Binding Assays- Reaction mixtures (20 g
u
4) as described previously (19). D-loop µl) contained indicated amounts of RECQ1 es
substrates were prepared as previously and 0.5 nM 32P-end-labeled DNA substrates t on
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described (13). in DNA binding buffer (20 mM Tris-HCl (pH o
v
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DNA Helicase Assays- Helicase reaction 7.5), 2 mM MgCl , 20 mM NaCl, 0.1 mg/ml m
2 b
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mixtures (20 µl) contained 20 mM Tris-HCl BSA, 1 mM dithiothreitol) containing 1 mM r 1
7
(pH 7.5), 10 mM KCl, 8 mM dithiothreitol, 5 ATPγS or no nucleotide. The reactions were , 2
0
mM MgCl2 and 5 mM ATP (unless otherwise incubated at 24°C for 15 min after addition of 18
indicated), 10% glycerol, 80 µg/ml BSA, 0.5 RECQ1. After incubation, 3 µl of loading dye
nM DNA substrate and the indicated RECQ1 (74% glycerol, 0.01% xylene cyanol and
concentrations. Reactions were initiated by bromophenol blue) was added to each
the addition of RECQ1 unless otherwise reaction, and samples were loaded on to native
noted, and the reaction mixtures were 5% (19:1 cross-linking ratio) polyacrylamide
incubated for the indicated times at 37oC. In gels, and electrophoresed at 200 V for 2.5 h at
kinetic experiments, 200 µl reactions 4°C using 1X TBE as the running buffer. The
containing forked duplex substrate (0.8 nM or resolved radiolabeled species were visualized
3.2 nM, as specified) and either 10 or 20 nM using a phosphorimager and analyzed with
RECQ1 were initiated with 5 mM ATP and 20 ImageQuant software (Molecular Dynamics).
µl aliquots were removed at 0-180 sec. Unless DNA Strand Annealing Assays- The DNA
otherwise indicated, helicase reactions were strand-annealing activity of RECQ1 was
terminated by the addition of 20 µl of Stop measured using partially or fully
Buffer (35 mM EDTA, 0.6% SDS, 25% complementary synthetic oligonucleotides
glycerol, 0.04% bromophenol blue, 0.04% (each at a concentration of 0.5 nM) of which
xylene cyanol) with a ten-fold molar excess of one was labeled at the 5'-end using [γ-32P]ATP
3
and T4 polynucleotide kinase. Annealing of a annealing activity as described above and
32P-labeled oligonucleotide (0.5 nM) to the other to assess the efficiency of
M13mp18 ssDNA (1 nM) was also measured. RECQ1 depletion by Western blotting
Strand annealing reactions (20 µl) were carried
analysis. In control experiments, Protein
out in helicase reaction buffer and contained
G agarose beads lacking any bound
the indicated RECQ1 concentrations. Where
antibody were incubated either with or
specified, RPA (0.19-96 nM), ESSB (0.75-768
without RECQ1 (20 nM), and the resulting
nM), ATP (2 or 5 mM), ATPγS (2 or 5 mM)
supernatants were tested for strand
and ADP (2 mM) were also present.
annealing.
Reactions were initiated by adding the
unlabeled DNA strand and were incubated for Partial Proteolytic Digestion of RECQ1-For
15 min at 37°C. In kinetic experiments, 200 the limited proteolysis studies, purified
µl reactions were initiated and 20 µl aliquots recombinant RECQ1 (2 µg) was incubated
were removed at 0.5, 1, 2, 4, 8, 16 and 32 min. with chymotrypsin (0.1 µg, Sigma) in the
A control reaction mixture was set up in an presence or absence of Mg+2 (3 mM) and ATP
identical manner to determine the level of or ATPγS (3 mM) in a 20 µl reaction mixture
spontaneous annealing of oligonucleotides in at 37°C for 45 min. An equal volume of 2X
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the absence of RECQ1. Reactions were SDS-sample buffer (126 mM Tris-HCl (pH ow
n
terminated by the addition of 20 µl of Stop 6.8), 20% glycerol, 4% SDS, 0.02% lo
a
Buffer (35 mM EDTA, 0.6% SDS, 25% bromophenol blue, 10% β-mercaptoethanol) de
d
glycerol, 0.04% bromophenol blue, 0.04% was added to the proteolytic digestion mixture fro
m
xylene cyanol). Reaction products were and samples were boiled for 5 min. Proteins h
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products were resolved on native 12% Coomassie stain. .jb
polyacrylamide gels. Radiolabeled DNA c.o
species in polyacrylamide gels were visualized RESULTS brg/
using a PhosphorImager and quantitated using y g
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ImageQuant software (Amersham Although the effect of duplex length es
t o
Biosciences). on RECQ1 helicase activity was determined n
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Immunodepletion- One microgram rabbit (15), we have characterized the unwinding ov
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polyclonal anti-RECQ1 antibody (Santa activity of purified recombinant RECQ1 m
b
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Cruz Biotech.) or normal rabbit IgG (Santa helicase on a variety of DNA substrates that r 1
7
Cruz Biotech.) was incubated for 1 h at are proposed intermediates of various , 2
0
4°C with 40 µl Protein G agarose beads pathways of cellular DNA metabolism. In 18
addition, we have characterized how RECQ1
(Roche Molecular Biochemicals) that had
acts upon unwound single-stranded DNA
been equilibrated in RECQ1 reaction
molecules to convert them to duplex strands
buffer (see above). Beads were
by a strand annealing activity.
subsequently washed three times with the
DNA Substrate Specificity of RECQ1
reaction buffer. Antibody-bound beads Helicase- To determine the DNA substrate
were then incubated with 20 nM RECQ1 preference for RECQ1 helicase activity, we
in reaction buffer (40 µl) for 1 h at 4°C. examined a series of B-form duplex DNA
Beads were collected by centrifugation substrates with a variety of combinations of
and the supernatant was again incubated single-stranded or double-stranded DNA tails
flanking the 19 bp duplex region. These DNA
with antibody-bound beads for 10 min at
substrates, characterized by a related
4°C for a second immunodepletion step.
nucleotide sequence, were incubated with
Following centrifugation, supernatants
increasing concentrations of purified
were collected and divided into two equal
recombinant RECQ1 helicase in the presence
aliquots, one to be assayed for strand
of ATP, and the reaction products were
4
analyzed on native polyacrylamide gels. The We next tested a forked duplex in
results, shown in Fig. 1, demonstrate that which both the 5’ and 3’ tails of the fork were
RECQ1 unwinds these substrates to different double-stranded. This three-way junction
extents depending on the presence and nature structure resembles a stalled replication fork
of the 3’ and 5’ tails. Of the substrates tested, where both the leading and the lagging strands
a forked duplex with 5’ and 3’ single-stranded have been converted to duplex DNA. As
DNA arms of 26 and 25 nt, respectively, was shown in Fig. 1B, the synthetic replication
unwound relatively efficiently (Fig. 1A, 1C). fork was unwound by RECQ1 in a protein
As expected from the unwinding polarity of concentration dependent manner. Quantitative
RECQ1 (15), a DNA substrate flanked by only analysis demonstrated that nearly the same
a 3’ ssDNA tail was also unwound by percentage of synthetic replication fork and 5’
RECQ1; however, significantly less flap substrate were unwound by RECQ1 at a
unwinding of this substrate was detected given helicase concentration, suggesting that
compared to the forked duplex with 3’ and 5’ the enzyme is indifferent to the single-
ssDNA arms throughout the RECQ1 protein stranded or double-stranded character of the 5’
titration. The greatest difference, 2.5-fold, arm of these substrates. Gel analysis of the
was observed at a RECQ1 concentration of 10 unwound products of the RECQ1 helicase
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nM (Fig. 1C). reaction with the synthetic replication fork o
w
n
The observation that RECQ1 revealed that the substrate was unwound in the lo
a
unwound a forked DNA duplex with direction of the fork, primarily yielding a de
d
noncomplementary 3’- and 5’-ssDNA tails product with the duplex arm intact (Fig. 1B). fro
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significantly better than the simple 3’ ssDNA These results suggest that RECQ1 recognized h
tailed duplex suggested that RECQ1, like and bound the junction and translocated in a ttp://w
WRN and BLM (18;19), might unwind other 3’-5’ direction along the bottom strand w
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DNA structures with junctions as helicase towards the direction of the fork thus .jb
substrates. Forked DNA substrates with either generating the partial duplex product. This c.o
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one or both of the arms in the double-stranded activity is similar to that observed for the b/
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state are intermediates of cellular processes WRN helicase (18), but is distinct from that of g
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including DNA replication, repair, and Drosophila RECQ5β which was shown to es
t o
recombination. We therefore tested this class unwind the lagging strand duplex of the n
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of substrates which include a 3’ flap, a 5’ flap, synthetic replication fork structure (20). o
v
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or a synthetic replication fork with 3’ and 5’ Importantly, the results suggest that RECQ1 m
b
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duplex arms. Interestingly, the 3’ flap unwinds conventional DNA duplex substrates r 1
7
substrate was unwound the best of all the that completely lack ssDNA character, and , 2
0
substrates tested. A significantly greater that its unwinding properties have both 18
percent of the 3’ flap substrate, as much as similarities and differences from other RecQ
3.5-fold, was unwound by RECQ1 compared helicases (see Discussion).
to the forked duplex with 3’ and 5’ ssDNA RECQ1 Helicase Activity Is Sensitive to Free
arms at the lowest RECQ1 protein Magnesium Cation- The overall duplex DNA
concentrations tested (0.6, 1.2, and 2.5 nM) unwinding reaction catalyzed by a helicase is
(Fig. 1C). A 5’ flap substrate was also an energy-requiring process and helicases
unwound by RECQ1, but not nearly to the utilize energy from the hydrolysis of ATP or
extent as either the forked duplex with ssDNA other nucleoside triphosphate (NTP), which
arms or the 3’ flap substrate. Despite the allows them to translocate and unwind DNA
absence of a 3’ ssDNA tail, RECQ1 unwound duplexes (1;2). As the ATP-Mg2+ complex is
the 5’ flap substrate as efficiently as the 3’ the source of chemical energy for the RECQ1
ssDNA tailed duplex, resulting in helicase-catalysed unwinding, we sought to
approximately 40% of the 5’ flap determine the optimal concentrations of Mg2+
oligonucleotide released at the highest RECQ1 and ATP for RECQ1 helicase activity.
concentration tested, 10 nM (Fig. 1C). Initially we varied the Mg2+ concentrations at
a fixed ATP concentration (5 or 1 mM) to
5
estimate the ratio between ATP and Mg2+ ATP concentration (Fig. 2B). RECQ1
where the greatest level of RECQ1 unwinding unwinding activity increased linearly with
could be detected, and then to determine the ATP concentration up to ~ 4-5 mM ATP
optimal ATP-Mg2+ concentration. As shown where a plateau was approached for either
in Fig. 2A, RECQ1 helicase activity in the fixed Mg2+: ATP ratio (Fig. 2B). A Michaelis-
presence of 5 mM ATP increased fairly Menten kinetic analysis to the initial
linearly with Mg2+: ATP ratios up to 0.5, but hyperbolic regime of the plot shown in Fig. 2B
decreased with greater Mg2+: ATP ratios, yielded K and V values of 0.99 mM and
m max
particularly evident at ratios greater than 1. A 5.63 X 10-4 nM sec-1, respectively, for the
steep decrease in RECQ1 helicase activity was 0.5:1 Mg2+: ATP ratio. For the 1:1 ratio of
observed at a Mg2+: ATP ratio of 1.25, Mg2+: ATP, the K and V values were 0.85
m max
resulting in two-fold reduction in the percent mM and 3.73 X 10-4 nM sec-1, respectively.
of forked duplex substrate unwound. Further Kinetics of RECQ1 Helicase Activity-Having
increases in the Mg2+: ATP ratio resulted in a established the optimal concentration of ATP
greater reduction of unwinding by RECQ1. and Mg2+ for the RECQ1 helicase reaction; we
At a Mg2+: ATP ratio of 2, only 10% of the examined the kinetics of unwinding on a
substrate molecules were unwound by RECQ1 forked duplex DNA substrate with 5’ and 3’
D
compared to 80% at a Mg2+: ATP ratio of 0.5. ssDNA arms using different RECQ1 protein o
w
To confirm the effect of free Mg2+ on RECQ1 or DNA substrate concentrations. Using a nlo
a
helicase activity, we carried out the same set fixed forked duplex substrate concentration of de
d
of experiments using 1 mM ATP, which also 0.8 nM, it was determined that the amount of fro
m
resulted in a steady increase in RECQ1 DNA substrate unwound by RECQ1 displayed h
helicase activity as a function of the Mg2+: a linear dependence with respect to time ttp://w
ATP ratio up to 0.5. Inhibition of RECQ1 throughout the 180 sec time course at helicase w
helicase activity at Mg2+: ATP ratios greater concentrations of 10 nM or 20 nM (Fig. 3A). w.jb
than 0.5 was observed; however, the decrease The initial rate of fork unwinding was c.o
rg
in RECQ1 unwinding did not display as great determined to be 0.0497 nM (bp)/s at a b/
a dependence on Mg2+: ATP ratio as observed RECQ1 concentration of 10 nM, whereas 20 y g
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at the 5 mM ATP concentration. These data nM RECQ1 unwound the fork duplex with an es
t o
indicate that RECQ1 helicase is sensitive to initial rate of 0.0735 nM (bp)/s. Thus, the n
N
the ratio of Mg2+ to ATP, with maximum initial rates of unwinding were dependent on o
v
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activity exhibited at Mg2+: ATP ratio of ~0.5- RECQ1 concentration. m
b
e
1.0. Beyond this optimum, the unwinding We next examined the kinetics of r 1
7
activity steadily declined with increasing RECQ1 helicase activity at a fixed RECQ1 , 2
0
Mg2+: ATP ratio suggesting that free Mg2+ ion concentration (20 nM), but at a four-fold 18
is inhibitory to RECQ1 helicase activity. greater concentration of forked duplex
Sensitivity to free Mg2+ ion was also reported substrate (3.2 nM). As shown in Fig. 3B, the
for the E. coli RecQ helicase (21); however amount of DNA substrate (3.2 nM forked
WRN helicase activity, which displayed duplex molecules) unwound by RECQ1
optimal unwinding at a Mg2+: ATP ratio of 1, exhibited linearity throughout the 180 sec time
was not adversely affected by greater Mg2+: course. For comparison, the time course from
ATP ratios up to 4 (22). Fig. 3A using the same concentration of
We next performed experiments to RECQ1 (20 nM) but only 0.8 nM forked
determine the optimal concentration of ATP- duplex is shown in Fig. 3B. A quantitative
Mg2+ complex for RECQ1 helicase activity. comparison of the rates of RECQ1 helicase
Since RECQ1 is inhibited by free Mg2+, activity at the two DNA substrate
RECQ1 helicase assays were performed in concentrations indicate a 2.7-fold increase
which the Mg2+-ATP complex, maintained at when 3.2 nM forked duplex substrate is
either a 1:1 or 0.5:1 ratio of Mg2+: ATP, was present in the reaction compared to 0.8 nM.
increased. The percent of forked DNA These kinetic results can be interpreted to
substrate unwound was plotted as a function of mean that optimal RECQ1 catalytic activity is
6
retained thoughout the 3 min time course the 3’ ssDNA tailed substrate may reflect its
under these reaction conditions and that at 0.8 DNA substrate binding affinity.
nM DNA substrate and 20 nM RECQ1, the RECQ1 Promotes Branch Fork Migration of
enzyme is not saturated with DNA substrate the Holliday Junction- The growing body of
since a significantly greater rate of unwinding evidence suggests that RecQ helicases are
was observed when a four-fold greater DNA involved in the replicational stress response by
substrate concentration was present in the their roles in a non-recombinogenic pathway
reaction. or RAD51-mediated homologous
DNA Substrate Binding by RECQ1-Although recombination (4;26;27). Localization of
DNA helicases, by their very nature, must load RecQ helicases (WRN, BLM, SGS1) at stalled
on to DNA substrates to initiate unwinding, DNA replication forks (25;28;29), and their
the relative abilities of RecQ helicases to ability to branch-migrate Holliday Junction
stably bind DNA molecules vary. For intermediates (24;25;30) distinguishes them
example, E. coli RecQ binds to and unwinds a from a number of other helicases involved in
large variety of duplex DNA substrates other aspects of DNA or RNA metabolism.
including blunt, ssDNA tailed, or forked To address a potential role of RECQ1 in
duplex molecules (23). SGS1 also binds a branch fork migration, we tested its helicase
D
number of oligonucleotide based duplex DNA activity on a synthetic four-stranded Holliday o
w
n
molecules to varying extents (24). On the Junction structure. As shown in Fig. 5A, lo
a
other hand, we and others have observed that RECQ1 unwound the HJ(X12) DNA molecule de
d
with the exception of the four-stranded in a protein concentration dependent manner. fro
m
Holliday Junction structure (25), full-length Eighty two percent of the HJ substrate was h
recombinant WRN helicase does not stably unwound by 10 nM RECQ1 in a 15 min ttp://w
bind to various oligonucleotide duplex DNA reaction. In these reactions, the major product w
w
substrates that the enzyme efficiently unwinds of HJ unwinding was splayed-arm product .jb
(18). Since the results presented here indicate suggesting RECQ1 catalyzed disruption of HJ c.o
rg
that RECQ1 helicase has the ability to unwind by branch fork migration. To address this b/
y
a large variety of DNA structures, we wanted possibility, we examined RECQ1 branch fork g
u
to characterize its physical interaction with migration activity of the HJ substrate that had es
t o
DNA molecules to better understand its been preincubated with increasing n
N
helicase substrate preference. Gel mobility concentrations of RuvA protein, an E. coli o
v
e
shift assays were used to measure DNA protein implicated in homologous m
b
e
binding by RECQ1 in the presence of the recombination that has a very high affinity to r 1
7
poorly hydrolysable analog ATPγS. As shown the Holliday Junction (31) and blocks WRN , 2
0
in Figs. 4A and 4B, RECQ1 effectively bound branch migration activity on α-structures (25). 18
either the forked duplex or 3’ ssDNA tailed RuvA inhibited RECQ1-catalyzed branch
duplex substrates, respectively, as evidenced migration in a RuvA concentration dependent
by the three distinct slowly migrating species manner (Fig. 5B). Consistent with its helicase
on native 5% polyacrylamide gels. Similar activity on the HJ, RECQ1 binds the HJ
patterns for gel-shifted species were observed substrate in a protein concentration dependent
for RECQ1 bound to ssDNA or blunt duplex manner as demonstrated by gel-shift
DNA (data not shown). The shifted species experiments (Fig. 5C). These results suggest
may possibly reflect DNA molecules bound that RECQ1 catalyzes branch migration of the
by distinct RECQ1 protein assembly states. synthetic HJ by initiating unwinding at the
Quantitative analysis of the DNA binding junction cross-over.
demonstrated that RECQ1 bound more RECQ1 Melts D-loops- The ability of
efficiently to the forked duplex compared to RECQ1 to unwind branched DNA structures
the 3’ ssDNA tailed duplex (Fig. 4A, 4B), and branch migrate a Holliday Junction
suggesting that the RECQ1 helicase substrate prompted us to examine if RECQ1 helicase
preference for the forked duplex compared to would metabolize other intermediates of
homologous recombination. An early step in
7
homologous recombination is the invasion of the D-loop with a 3’ protruding single
single stranded DNA into a homologous stranded tail compared to the D-loop
duplex to form a D-loop structure in which substrates with a third strand that has a 5’
one strand of a duplex DNA molecule is ssDNA tail or no tail at all (Fig. 6D). In a 32
displaced concomitant with formation of min time course, RECQ1 catalyzed release of
Watson-Crick base pairing between the 89%, 63%, or 37% of the invading third strand
complementary strand and an incoming single from the D-loops with 3’ protruding single
strand (32-34). To test RECQ1 activity on strand tail, 5’ protruding single strand, or no
these early recombination intermediates, we tail, respectively (Fig. 6D). For comparison,
prepared the D-loop substrates by annealing we examined the ability of RECQ1 to unwind
three oligonucleotides designed to form a a bubble substrate in which the top and bottom
single molecule (61 nucleotide residues in strands were the same as those used for the
length) with a central noncomplementary creation of the three-stranded D-loop
bubble of 21 residues flanked by 20 nucleotide structures. Only 9% of the bubble was
residues of duplex to either side and in which unwound by RECQ1 in 32 min (Supp Data
the third strand invades and base pairs with Fig. 1), suggesting that RECQ1 preferentially
one of the looped out strands in the 21 unwinds the three stranded D-loop structures
D
nucleotide bubble region (13). We first compared to a bubble substrate. Despite the o
w
n
examined the effect of RECQ1 on D-loops observed differences in rates of unwinding lo
a
with protruding 5' or 3' single-stranded tails of between the D-loop and bubble structures, de
d
20 residues extending from the invading third RECQ1 was able to bind both classes of DNA fro
m
strand. In each case, the invading third strand molecules similarly, as judged by gel mobility h
was 5’ 32P-labeled so that dissociation of this shift assays (Supp. Data Fig. 2). Thus ttp://w
strand could be monitored on native preferential unwinding of the D-loop substrate w
w
polyacrylamide gels. RECQ1 was able to by RECQ1 is not determined solely by the .jb
unwind D-loops with either 3’ (Fig. 6A) or 5’ enzyme’s DNA binding ability. c.o
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(Fig. 6B) protruding single stranded tail to RECQ1 Catalyzes DNA Strand Annealing- b/
y
release the invading third strand in a protein Intermediates in homologous recombination g
u
concentration dependent manner; however, are believed to involve multi-stranded es
t o
preferential unwinding of the D-loop with a 3’ branched recombinant DNA molecules that n
N
protruding single stranded tail was observed are a consequence of strand invasion and o
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over the entire range of RECQ1 protein processing during Holliday Junction m
b
e
concentrations (Fig. 6A-6C). For example, at resolution. Our observations that RECQ1 has r 1
7
2.5 nM RECQ1 ~16% of the invading third the ability to stably bind DNA molecules , 2
0
strand with a 3’ tail was released from the D- raised the possibility that its intrinsic DNA 18
loop compared to only 6% of the 5’ tailed binding activity might be utilized for functions
third strand (Fig. 6C); at 20 nM RECQ1, other than duplex DNA unwinding fueled by
approximately 88% and 51% of the third ATP hydrolysis. The recent report that human
strands were released from the D-loop with 3’ RECQ5β catalyzes strand annealing (16)
and 5’ protruding single-stranded tails, raised the possibility that RECQ1 might also
respectively (Fig. 6C). perform this activity. Although other RecQ
We next performed kinetic analyses of D- helicases have not been reported to catalyze
loop unwinding by RECQ1. In addition to D- strand annealing, specialization of human
loops with protruding 5' or 3' single-stranded RecQ helicase function may be at least partly
tails, we examined a D-loop structure with achieved by its coordinate balance between
flush ends in which the third strand was unwinding and strand annealing activities.
completely complementary to the internal 21 To investigate a putative RECQ1
nt bubble region nucleotides. Release of the strand annealing activity, we incubated two
invading third strand was monitored over a 32 partially complementary single strand
min time course (Fig. 6D). RECQ1 (10 nM) oligonucleotides (0.5 nM each) with
displayed a strong preference for unwinding increasing concentrations of purified
8
recombinant RECQ1 for 15 min and analyzed specificity of inhibition of strand annealing by
the products on native polyacrylamide gels. If RECQ1, we tested the effect of either RECQ1
strand annealing transpired, a 19 bp forked antibody in reactions containing BLM
duplex would be formed. The results from a recombinant protein, which was shown to
typical experiment, shown in Fig. 7A, catalyze strand annealing (C. F. Cheok and I.
demonstrate that RECQ1 promotes strand D. Hickson, personal communication). Either
annealing of the two complementary strands to RECQ1 antibody did not inhibit strand
form the splayed arm duplex which co- annealing catalyzed by BLM (data not shown).
migrates with the intact forked duplex control. To provide further evidence that RECQ1 is
RECQ1 promoted strand annealing was responsible for the observed strand annealing,
dependent on RECQ1 concentration with an immunodepletion experiments were
increasing amount of the annealed product performed. In these studies RECQ1 was
begin formed up to 20 nM RECQ1; however, immunodepleted prior to the strand annealing
higher RECQ1 concentrations greater than 20 reaction. As shown in Fig. 8B, RECQ1 strand
nM inhibited strand annealing (Fig. 7A, 7B). annealing was greatly reduced in the reaction
For comparison, RECQ1 helicase activity on that was immunodepleted for RECQ1 (lanes 2
the forked duplex substrate over the same versus 4). Western blot analysis confirmed
D
protein concentration range under the same that RECQ1 protein was significantly depleted o
w
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reaction conditions (with the exception that 5 by its pretreatment with the RECQ1 antibody lo
a
mM ATP is present in the helicase reactions) (Fig. 8C, lane 3). In control reactions, de
d
is shown (Fig. 7B). This comparison preincubation of RECQ1 protein with normal fro
m
demonstrates that RECQ1-catalyzed strand IgG did not have an inhibitory effect on h
annealing and helicase activities display a very RECQ1 strand annealing (Fig. 8B, lane 3) and ttp://w
similar dependence on RECQ1 concentration failed to immunodeplete RECQ1 (Fig. 8C, w
w
until the plateau is reached. These results lane 2). These results provide evidence that .jb
suggest that RECQ1 has comparable ability to RECQ1 was responsible for catalyzing strand c.o
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unwind the forked duplex substrate (in the annealing since the activity was inhibited in a b/
y
presence of ATP) or catalyze annealing of specific manner by two RECQ1-directed g
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complementary single strands at low RECQ1 antibodies, one against a unique peptide es
t o
concentrations. A kinetic analysis sequence and the other against the N-terminus n
N
demonstrated that strand annealing catalyzed of RECQ1. o
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by an optimal RECQ1 concentration (20 nM) We next tested the ability of RECQ1 m
b
e
displayed an initial linear rate of strand to catalyze strand annealing of a 100-mer to a r 1
7
annealing during the first 8 min of the target sequence in the M13mp18 ssDNA , 2
0
reaction, resulting in ~52% of the single circular molecule. Although RECQ1 18
stranded DNA converted to forked duplex promoted annealing of the 100-mer to the
(Fig. 7C). RECQ1 promoted 75% of the M13mp18 ssDNA, a higher RECQ1 protein
single-stranded molecules to anneal by 32 min concentration was required to obtain the 100
compared to ~16% spontaneous annealing that bp M13 partial duplex product and the yield of
occurred in the absence of RECQ1 (Fig. 7D). the annealed product was very low, ~14% of
To address if RECQ1 is responsible the product formed by 160 nM RECQ1 in a 15
for the observed strand annealing activity, we min assay (Supp. Data Fig. 3). These results
examined the effect of RECQ1 antibody on suggest that RECQ1 promoted strand
RECQ1-catalyzed strand annealing. annealing is compromised by the presence of
Preincubation of RECQ1 with increasing large heterologous sequence in the DNA
amounts of a RECQ1 peptide antibody (14) substrate, and is dependent on DNA
resulted in the inhibition of RECQ1 strand homology.
annealing (Fig. 8A, lanes 2-5 versus lane 7). Modulation of RECQ1 Strand Annealing by
Similar inhibition was observed using an Nucleotide Status- The ability of RECQ1 to
antibody directed against the N-terminus of catalyze DNA unwinding or strand annealing
RECQ1 (data not shown). To address the depending on ATP status suggested that the
9
strand annealing activity might be regulated role in the modulation of RECQ1 strand
by nucleotide binding and/or hydrolysis. To annealing. To address this, RECQ1 strand
address this issue, RECQ1 strand annealing annealing to form the blunt duplex DNA
was performed in the presence of 2 mM ATP, product was conducted in the presence of 5
ADP, or the poorly hydrolysable ATP analog mM ATP or ATPγS. At 5 mM ATP, a slight
ATPγS. At an optimal RECQ1 concentration but statistically significant greater inhibition of
for strand annealing, 20 nM, RECQ1 strand annealing was observed compared to 2
converted 76% of the partially complementary mM ATP (Fig. 9C, 9D). However, at 5 mM
single strand oligonucleotide (0.5 nM) into ATPγS, significantly greater inhibition of
forked duplex product in the absence of any strand annealing was detected compared to 2
nucleotide (Fig. 9A, lane 2; 9B); in contrast, mM ATPγS. In fact, the level of RECQ1
~36% annealed product was obtained in the strand annealing in the presence of 5 mM
presence of ATP (Fig. 9A, lane 3;9B). The ATPγS was very comparable to spontaneous
presence of ATPγS inhibited the strand strand annealing (29%) (Fig. 9C, 9D). These
annealing further to ~20% (Fig. 9A, lane 4; results suggest that ATP-binding by RECQ1 is
9B), whereas the presence of ADP did not inhibitory to its strand annealing activity.
have any significant negative effect on To gain further insight to the effect of
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RECQ1 strand annealing (Fig. 9A, lane 5; ATP on RECQ1 strand annealing, we tested a o
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9B). mutant RECQ1 recombinant protein lo
a
As the forked duplex product of the containing a site-specific replacement of the de
d
strand annealing reaction is a very good conserved lysine residue of the Walker A box fro
m
substrate for RECQ1 helicase activity, it was (motif I) implicated in ATP binding with an h
possible that the observed inhibition of alanine residue (RECQ1-K119A). The ttp://w
RECQ1 mediated strand annealing in the K119A RECQ1 mutation abolished RECQ1 w
w
presence of ATP may be due to a competition helicase activity on the same forked duplex .jb
between the helicase and strand annealing substrate used in this study (17) and also c.o
rg
activities of the protein. Therefore, we eliminated RECQ1 ATPase activity (data not b/
y
performed similar experiments using 44-mer shown). As shown in Fig. 9E, the RECQ1- g
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complementary single strand oligonucleotides K119A mutant protein retained its ability to es
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that will generate a 44 bp blunt duplex product catalyze strand annealing comparable to wild- n
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upon annealing that will not be unwound by type RECQ1 protein. However, in the o
v
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RECQ1 helicase in the presence of ATP. As presence of ATPγS, RECQ1-K119A, unlike m
b
e
shown in Fig. 9C, RECQ1 (20 nM) catalyzed wild-type RECQ1, retained its ability to r 1
7
efficient annealing of the complementary catalyze strand annealing. These results , 2
0
single strands such that 93% of the single- provide further evidence that the observed 18
stranded complementary oligonucleotides strand annealing activity is intrinsic to RECQ1
were converted to blunt duplex product in the and support our findings that ATP binding by
absence of ATP (Fig. 9C, lane 2). The RECQ1 alters the protein’s ability to perform
presence of 2 mM ATP resulted in a 17% strand annealing.
reduction in RECQ1-mediated strand We next wanted to examine if ATPγS
annealing (Fig. 9C, lane 3), whereas a 28% modulated RECQ1 binding to single- or
reduction was obtained when the strand double stranded DNA to determine if this was
annealing was performed in the presence of 2 a component of the nucleotide’s inhibition of
mM ATPγS (Fig. 9C, lane 4). ADP (2 mM) RECQ1 strand annealing. RECQ1 bound
did not affect RECQ1 mediated strand efficiently to a single-stranded 44-mer or a 44
annealing (Fig. 9C, lane 5). bp duplex in the presence or absence of 2 mM
Since recombinant RECQ1 was ATPγS (Fig. 10A). Under conditions in which
shown to be a DNA dependent ATPase (14) RECQ1 was pre-bound separately to the two
and can also hydrolyze ATP in the presence of complementary oligonucleotides, significantly
double stranded DNA (our unpublished data), reduced strand annealing was observed when
it was conceivable that ATP turnover played a ATPγS was present (Fig. 10B), as observed
10