in a HSP90ATPgS/AGO1/siRNA duplex
complex. In contrast, completing RISC
assembly in the presence of ATP showed
that AGO1 was mostly loaded with single-
tions, Iki and colleagues suggested the
following model for plant HSP90-facili-
and subsequent binding of ATP causes
conformational changes in HSP90 and
AGO1 that allow siRNA duplexes to bind
to the AGO1. The hydrolysis of ATP by the
HSP90 ATPase activity causes the dis-
lowed by an additional conformational
rearrangement in AGO1 that could facili-
tate the removal of the passenger strand.
Intriguingly, the ‘‘rubber band’’ model
for RISC assembly in animals differs
from the proposed plant RISC assembly
pathway in a key way: it suggests that
ATP hydrolysis by HSP90 is required
reflects an HSP90-dependent conforma-
tional change in AGO proteins that allows
them to receive siRNA duplexes. In the
plant system, the formation of a stable
HSP90ATPgS, AGO1, and siRNA duplex
complex argues against the need for
ATP hydrolysis at this step. It remains to
be determined whether the use of ATPgS
in the animal RISC assembly system
would result in RNA duplex binding to the
HSP90ATPgS/AGO complex, which would
converge the plant and animal RISC
Taken together, the results presented
by Iki et al. (2010) and Iwasaki et al.
(2010) indicate that RISC loading with
small RNA duplexes in plants and animals
is ATP driven and requires the HSP70/
HSP90 chaperone machinery. The data
raise many intriguing mechanistic and
functional questions: What molecular
contacts and structural rearrangements
of HSP90 and AGO occur during RISC
assembly? How does HSP90 influence
the interaction of AGO and Dicer (Tahbaz
et al., 2004)? Is the ATPase activity modu-
lated by specific cochaperones that are
needed for HSP90 action during RISC
formation? How does the separation of
miRNA/miRNA* and siRNA duplexes dif-
fer in tobacco AGO1-containing com-
plexes? The development of a plant RISC
assembly system, in particular, will help
shed light on these questions and deepen
our understanding of RISC assembly in
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Repair Scaffolding Reaches New
Heights at Blocked Replication Forks
Michael Downey,1Ellen R. Edenberg,1and David P. Toczyski1,*
1Department of Biochemistry and Biophysics, University of California, San Francisco, 1450 3rd Street, San Francisco, CA 94158-9001, USA
In this issue of Molecular Cell, Ohouo et al. (2010) show that Mec1 (hATR) promotes the association of Slx4
and Rtt107 with Dpb11 (hTopBP1) in response to MMS-induced DNA alkylation, suggesting that Slx4 and
Rtt107 might coordinate repair factors specifically at damaged replication forks.
response focused on the relatively simple
case of double-strand breaks. Lesions
encountered during replication require a
much more complicated series of events
in which a cell must delay fork progres-
studiesoftheDNAdamagetion, and subsequently resume DNA
synthesis—all without disrupting the deli-
cate fork structure. This reorganization
requires the recruitment of several repair
and signaling complexes, in part facili-
domain-containing proteins. BRCT do-
mains recognize phosphorylated targets,
in many cases mediated by the DNA
damage-responsive kinase Mec1 (hATR).
Several proteins important for resistance
to replication stress, such as the yeast
proteins Dpb11 (hTopBP1) and Rtt107,
have several pairs of BRCT domains and
Molecular Cell 39, July 30, 2010 ª2010 Elsevier Inc.