Novel pro- and anti-recombination
activities of the Bloom’s
Dmitry V. Bugreev,1,2Xiong Yu,3Edward H. Egelman,3and Alexander V. Mazin1,4
1Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania
19102, USA;2Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Science,
Novosibirsk 630090, Russia;3Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences
Center, Charlottesville, Virginia 22908, USA
Bloom’s syndrome (BS) is an autosomal recessive disorder characterized by a strong cancer predisposition. The
defining feature of BS is extreme genome instability. The gene mutated in Bloom’s syndrome, BLM, encodes a
DNA helicase (BLM) of the RecQ family. BLM plays a role in homologous recombination; however, its exact
function remains controversial. Mutations in the BLM cause hyperrecombination between sister chromatids
and homologous chromosomes, indicating an anti-recombination role. Conversely, other data show that BLM
is required for recombination. It was previously shown that in vitro BLM helicase promotes disruption of
recombination intermediates, regression of stalled replication forks, and dissolution of double Holliday
junctions. Here, we demonstrate two novel activities of BLM: disruption of the Rad51-ssDNA (single-stranded
DNA) filament, an active species that promotes homologous recombination, and stimulation of DNA repair
synthesis. Using in vitro reconstitution reactions, we analyzed how different biochemical activities of BLM
contribute to its functions in homologous recombination.
[Keywords: Bloom’s syndrome; BLM helicase; homologous recombination; Rad51; RecQ; Rad54]
Supplemental material is available at http://www.genesdev.org.
Received August 24, 2007; revised version accepted October 2, 2007.
In humans, mutations in the BLM gene cause Bloom’s
syndrome (BS), an autosomal recessive disorder whose
clinical manifestations include proportional dwarfism,
immunodeficiency, male infertility, and others (German
1993). A specific feature of BS is a greatly elevated inci-
dence of cancer with exceptionally early onset. BS indi-
viduals are predisposed to develop most types of cancer,
an unusual feature among cancer predisposition disor-
ders. The hallmark of BS is a high degree of genome
instability (Bachrati and Hickson 2003; Hickson 2003).
Specifically, BS shows a greatly increased frequency of
reciprocal sister chromatid exchanges (SCE) and ex-
changes between homologous chromosomes (German
1993). These data indicate that BLM suppresses homolo-
gous recombination (HR), the process responsible for
chromosomal exchanges (Sonoda et al. 1999; Gonzalez-
Barrera et al. 2003). However, recent genetic data re-
vealed a more complex relationship between BLM and
HR: BLM may suppress some recombination events and
promote others (Adams et al. 2003).
In eukaryotes, HR has several crucial functions, in-
cluding segregation of homologous chromosomes, propa-
gation of genetic diversity, maintenance of telomeres,
and repair of double-stranded DNA breaks (DSB) (Hoeij-
makers 2001; West 2003; Krogh and Symington 2004;
Whitby 2005; Agarwal et al. 2006; Sung and Klein 2006;
Helleday et al. 2007). HR is performed by an assembly of
specialized proteins, in which Rad51 plays a central role
(Bianco et al. 1998). Rad51 forms a nucleoprotein fila-
ment on single-stranded DNA (ssDNA), which is gener-
ated by specialized exonucleases at the sites of DNA
breaks. The filament possesses unique activities; it per-
forms the search for homologous double-stranded DNA
(dsDNA) sequences and promotes subsequent DNA
strand exchange between ssDNA and homologous
dsDNA sequences (Sung et al. 2003). DNA strand ex-
change results in formation of joint molecules (D-loops),
in which the invading ssDNA serves as a primer and the
homologous dsDNA as a template for DNA polymerase
during DNA repair synthesis. The joint molecules con-
tinue down one of the two pathways (Allers and Lichten
2001; Hunter and Kleckner 2001). They either dissociate,
leading to rejoining of the broken chromosome through
synthesis-dependent strand annealing (SDSA) (Allers and
Lichten 2001), or proceed through the capture of the sec-
ond processed DNA end to produce Holliday junctions
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GENES & DEVELOPMENT 21:3085–3094 © 2007 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/07; www.genesdev.org3085
(Schwacha and Kleckner 1995; Cromie et al. 2006),
which are later resolved by structure-specific endonucle-
ases to produce crossover and noncrossover recombi-
nants via the DSB repair (DSBR) mechanism (Pâques and
Haber 1999). Whereas crossing over is essential for
proper chromosome disjunction during meiosis, it may
be detrimental during mitotic recombination due to loss
of heterozygosity (LOH) and genome rearrangements.
Consequently, mitotic recombination proceeds mainly
through the SDSA mechanism producing noncrossover
The product of the BLM gene is BLM helicase, a mem-
ber of the highly conserved RecQ family, which is re-
sponsible for genome maintenance in all organisms from
bacteria to humans (Wu and Hickson 2006). Consistent
with its role in HR, BLM physically interacts with HR
proteins Rad51 and Rad51D, as well as with several
other proteins involved in DNA repair and DNA-damage
signaling such as Mus81, MLH1, RPA, and ATM
(Beamish et al. 2002; Sharma et al. 2006). BLM can also
specifically recognize Holliday junctions and promote
their branch migration in an ATPase-dependent manner
(Karow et al. 2000). In vitro, BLM was shown to unwind
D-loops (van Brabant et al. 2000; Bachrati et al. 2006),
catalyze regression of model replication forks (Ralf et al.
2006), and resolve double Holliday junctions (Wu and
Hickson 2003) by forming a complex with topoisomerase
III? (Topo III?) and BLAP75 (Raynard et al. 2006; Wu et
Here we identified two novel biochemical activities of
BLM. We found that it can disrupt the Rad51-ssDNA
filament by dislodging human Rad51 (hRad51) protein
from ssDNA in an ATPase-dependent manner, the activ-
ity consistent with suppression of HR at an early stage.
We also demonstrated that BLM can stimulate DNA re-
pair synthesis and thereby possibly promote HR at the
late stages. Using in vitro reconstitution of HR reactions
we analyzed how the biochemical activities of BLM can
either promote or inhibit the SDSA mechanism of HR at
BLM inhibits DNA strand exchange activity of Rad51
BLM helicase shares similarity in its biochemical prop-
erties with the yeast Srs2 helicase, which is known to
dissociate the Rad51 filament (Krejci et al. 2003; Veaute
et al. 2003). In addition, overexpression of Sgs1, the sole
yeast RecQ ortholog, can suppress recombination defects
of srs2?, indicating at least partial overlap in their func-
tions (Mankouri et al. 2002; Ira et al. 2003). We hypoth-
esized that BLM can dissociate the filament formed by
hRad51 on ssDNA, in the same manner as Srs2. Here we
tested this hypothesis.
Since filament disruption would result in inhibition of
DNA strand exchange, we first used the D-loop assay to
test the effect of BLM helicase on DNA strand exchange
promoted by hRad51. In this assay, hRad51 forms a nu-
cleoprotein filament on ssDNA, which then catalyzes
formation of joint molecules (D-loops) with pUC19 su-
percoiled DNA (scDNA) (Fig. 1A). The reaction also con-
tained human RPA (hRPA), a ubiquitous eukaryotic
ssDNA-binding protein involved in DNA repair, replica-
tion, and HR, which was added after hRad51-ssDNA fila-
ment formation. The presence of hRPA was expected to
facilitate detection of hRad51 displacement from ssDNA
by BLM: Previously, it was shown that yeast RPA by
efficient binding to ssDNA prevents rapid reassembly of
the yeast Rad51-ssDNA filament after its disruption by
Srs2 (Krejci et al. 2003). As we showed previously, for-
mation of an active hRad51-ssDNA filament requires
Ca2+(Bugreev and Mazin 2004). Therefore, to activate
the hRad51-ssDNA filament, which was initially as-
sembled in the presence of Mg2+alone, calcium (2 mM)
was added to the reaction mixture prior to addition of
pUC19 scDNA. As expected, hRad51 efficiently pro-
moted formation of D-loops (Fig. 1B, lane 1). However,
addition of BLM to the hRad51-ssDNA filament prior to
Ca2+resulted in a strong inhibition of D-loop formation
(Fig. 1B, lane 2). In contrast, we did not observe a signifi-
cant inhibition when BLM was added to the filament
after addition of Ca2+(Fig. 1B, lane 7). The effect of the
order of addition of BLM relative to Ca2+indicated that
the inhibition was mediated by BLM interaction with an
inactive hRad51-ssDNA filament, presumably causing
its disruption, not by melting of the D-loops. Ca2+might
prevent dislodging of hRad51 from ssDNA by BLM be-
cause it stabilizes the filament (Bugreev and Mazin
2004). The inhibition of D-loop formation showed de-
pendence on the BLM concentration, with a half-inhibi-
tion (IC50) of ∼40 nM BLM (Fig. 1D,E). This BLM con-
centration was ∼75-fold lower than that of hRad51, indi-
cating that BLM acts catalytically; e.g., by translocating
along ssDNA. Consistent with this interpretation, the
ATPase-deficient BLM mutant, K695R, which lacks
DNA translocation and helicase activities, did not in-
hibit D-loop formation (Fig. 1B, lane 3). We found that
inhibition of D-loop formation was specific for hRad51;
BLM did not significantly inhibit DNA strand exchange
activity of hDmc1, a meiotic hRad51 homolog (Fig. 1B
[lanes 4,5], C), or yeast Rad51 protein (Fig. 1D, lanes
8–12). Conversely, inhibition of DNA pairing activity of
hRad51 appeared to be specific for BLM, as RecQ1, an-
other member of the human RecQ helicase family, in
concentrations from 25 to 200 nM did not significantly
inhibit Rad51-mediated D-loop formation under the
tested conditions (data not shown). Overall, the data pre-
sented here suggest that the observed inhibition of D-
loop formation is caused by disruption of the hRad51-
ssDNA filament by BLM helicase.
BLM dismantles the hRad51-ssDNA filament
To demonstrate that BLM indeed displaces hRad51 from
ssDNA, we developed a nuclease protection assay (Fig.
2A). Because hRad51 can bind both ssDNA and dsDNA,
32P-labeled dsDNA fragment was used as a trap for
hRad51 that would be displaced from the filament. The
dsDNA fragment contained a unique DdeI site, such that
binding of hRad51 would protect the dsDNA from cleav-
Bugreev et al.
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