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.
coordinate multiple binding
partners at sites of damage.
In this issue, Ohouo et al.
(2010) use mass spectrom-
etry to expand the repertoire
of proteins associated with
these scaffolds in damage-
treated cells. They show that
as well as the Slx4, Rtt101,
and Smc5/Smc6 complexes.
Ohouo et al. (2010) identify
SILAC to quantitatively com-
pare proteins recovered in
paired Rtt107 immunoprecip-
itates. Specifically, they iden-
tify a Mec1-dependent inter-
action between Dpb11 and
Rtt107 (Figure 1). During un-
perturbed DNA replication,
Dpb11 has a separate, es-
sential function in origin firing
in which it simultaneously
binds two replication factors
through its BRCT domains.
In response to DNA damage,
Dpb11 is recruited to stalled
replication forks and is now linked for
the first time to Rtt107 and Slx4 (Ohouo
et al., 2010). Both Rtt107 and Slx4 have
previously been implicated in replication
fork restart following transient exposure
to DNA-damaging agents (Rouse, 2004;
Roberts et al., 2006). Slx4 itself is thought
of as a scaffold for multiple structure-
specific endonuclease complexes, in-
cluding Slx1 and Rad1/Rad10 (Fricke
and Brill, 2003; Flott et al., 2007). In addi-
tion, Slx4 has previously been shown to
be required for Rtt107 phosphorylation
in response to DNA damage, although
the function of Rtt107 phosphorylation is
debated (Rouse, 2004; Roberts et al.,
2006; Ohouo et al., 2010). Here, the
authors show that Mec1 phosphorylation
of Slx4 is required for its binding to
Dpb11 (Ohouo et al., 2010). This phos-
phorylation is likely to mediate a direct
interaction with some of Dpb11’s BRCT
domains, although confirmation of this will
await further analysis. In addition, deletion
of the gene encoding the downstream
kinase Rad53 (hChk2) also eliminates
tion, hinting at more complicated higher-
order interactions (Ohouo et al., 2010).
Despite the fact that the N terminus of
Rtt107, containing four of its six BRCT
domains, is necessary and sufficient for its
interaction with Slx4, this interaction does
not appear to require DNA damage or the
checkpoint pathway (Roberts et al., 2006;
Ohouo et al., 2010). This suggests that the
interaction is mediated by a kinase that is
domain adjacent to the N-terminal BRCTs
mediates binding to Slx4.
Important clues to the function of the
Rtt107 scaffold in replication fork repair
come from the observation that it binds
the Rtt101 and Smc5/Smc6 complexes
(Ohouo et al., 2010; Roberts et al., 2008).
The Rtt101 cullin and its binding partners,
tin ligase complex whose substrates are
unknown (Zaidi et al., 2008). Rtt101 may
ubiquitinate a protein whose destruction
by the proteasome is required for fork
restart,or it may assemble ubiquitin chains
that contribute to existing scaffold struc-
tures at sites of damage. In mammalian
cells, RNF8 and RNF168 ubiquitin ligases
collaborate to attach ubiquitin chains onto
histones at DNA lesions, which recruit
and Durocher, 2009). RNF168
recruitment relies on prior
sumoylation at sites of DNA
damage (Galanty et al., 2009).
To this end, the Smc5/Smc6
complex shown to interact
with Rtt107 in this study in-
the substrates of Mms21 and
Rtt101 will reveal how these
proteins aid in repairat broken
One question arising from
Rtt107 scaffold is temporally
or spatially remodeled during
the course of a repair event
and whether its binding part-
Because each tandem BRCT
domain likely interacts with
one phosphoepitope at a time,
it is possible that the BRCT
domains of the scaffold se-
quentially bind and exchange
et al. (2010) provide evidence
tion between Dpb11 and Rtt107 is par-
ticularly important for resistance to MMS,
consistent with the fact that Slx4 mutants
are particularly sensitive to this damage
tive to a wide array of DNA-damaging
agents (Roberts et al., 2008). Whereas the
Slx4/Rtt107 complex binds Dpb11 after
treatment with MMS, binding of Rtt107 to
chromatin depends on both Rtt101 and
the Rtt109 histone acetyltransferase (Rob-
erts et al., 2008). Perhaps, different com-
plexes are assembled following treatment
actions with Rtt107 and the Rad1/Rad10
nuclease appear to be mutually exclusive,
suggesting that these associations may
also be context dependent (Flott et al.,
homolog in S. pombe, binds to the
(Williams et al., 2010). Whether this inter-
action is conserved in budding yeast
remains to be tested. A sampling of the
Rtt107 interactome at different times
during the course of repair or following
treatment with different damaging agents
will shed important light on the plasticity
of repair scaffolds.
Figure 1. A Model for Scaffolding at a Stalled Replication Fork
The BRCT scaffold Rtt107 and the Cul4-related Rtt101 become chromatin
by damage (blue star). The recruitment of both complexes depends upon the
HAT Rtt109, which has established roles in both unperturbed replication and
upon replication stress. The S. pombe Rtt107 homolog Brc1 has been shown
to associate with the Mec1 target H2A(X). Here, the authors show that Mec1
(hATR) phosphorylation of Slx4 promotes an association with the BRCT scaf-
fold Dpb11. This association also requires the presence of the Rtt107 scaffold.
Slx4 itself is thought to target many structure-specific nucleases to their sites
Sm6 complex, which contains a sumo ligase. Touching proteins represent
binding dependencies, but not necessarily direct interactions.
Molecular Cell 39, July 30, 2010 ª2010 Elsevier Inc.
Finally, the description of Rtt107, Slx4,
and Dpb11 as scaffold proteins that func-
tion solely to recruit downstream factors
may be an oversimplification. Dpb11
(TopBP1) and Slx4 have demonstrated
roles in the direct activation of Mec1
respectively (Kumagai et al., 2006; Fricke
and Brill, 2003). Perhaps there are also
additional, catalytic functions of Rtt107
yet to be described.
Flott, S., Alabert, C., Toh, G.W., Toth, R., Suga-
wara, N., Campbell, D.G., Haber, J.E., Pasero, P.,
and Rouse, J. (2007). Mol. Cell. Biol. 27, 6433–
Fricke, W.M., and Brill, S.J. (2003). Genes Dev. 17,
Galanty, Y., Belotserkovskaya, R., Coates, J.,
Polo, S., Miller, K.M., and Jackson, S.P. (2009).
Nature 462, 935–939.
Kumagai, A., Lee, J., Yoo, H.Y., and Dunphy, W.G.
(2006). Cell 124, 943–955.
Ohouo, P.Y., Bastos de Oliveira, F.M., Almeida,
B.S., and Smolka, M.B. (2010). Mol. Cell 39, this
Panier, S., and Durocher, D. (2009). DNA Repair
(Amst.) 8, 436–443.
Roberts, T.M., Kobor, M.S., Bastin-Shanower,
S.A., Ii, M., Horte, S.A., Gin, J.W., Emili, A., Rine,
J., Brill, S.J., and Brown, G.W. (2006). Mol. Biol.
Cell 17, 539–548.
Roberts, T.M., Zaidi, I.W., Vaisica, J.A., Peter, M.,
and Brown, G.W. (2008). Mol. Biol. Cell 19, 171–
Rouse, J. (2004). EMBO J. 23, 1188–1197.
Guenther, G., Tainer, J.A., and Russell, P. (2010).
EMBO J. 29, 1136–1148.
J.S., Williams,R.S., Dovey,C.L.,
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Dangerous Liaisons: Fanconi Anemia
and Toxic Nonhomologous End Joining
in DNA Crosslink Repair
Samuel F. Bunting1and Andre ´ Nussenzweig1,*
1Experimental Immunology Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA
The proper choice of repair pathway is critical to tolerating various types of DNA damage. In a recent issue of
Molecular Cell, Adamo et al. (2010), along with a second report (Pace et al., 2010), describe how the Fanconi
icant new opportunity for the treatment of FA.
In 1927, the Swiss pediatrician Guido
Fanconi described a fatal progressive
anemia that had caused the deaths of
three brothers. This inherited disease
came to be known as Fanconi’s anemia
(FA) and is associated with congenital
abnormalities, failure of hematopoiesis,
and a high predisposition to cancer.
Over eighty years later, our knowledge
of the genetic basisof FA has progressed,
but our ability to treat affected individuals
is still limited. FA is classified into 13
subtypes according to the presence of
homozygous mutations in any of 13
known FANC genes. Eight of the FANC
genes encode factors that make up the
FA ‘‘core complex’’ (FANCA-C, E-G, L,
and M), which catalyzes the monoubiqui-
tylation and activation of the FANCD2 and
FANCI proteins. Ubiquitylated FANCD2
and FANCI are recruited to chromatin,
where they facilitate DNA repair. Three
factors that are associated with the
homologous recombination (HR) DSB
repair pathway—FANCD1, FANCN, and
FANCJ—act downstream of FANCD2-
Cells from FA patients of all sub-
types exhibit chromosome abnormalities
when treated with DNA crosslinking
agents such as mitomycin C (Moldovan
D’Andrea, 2004), which block replication
and transcription. Defective interstrand
underlie the clinical and cellular pheno-
types associated with FA. DNA crosslink
processing utilizes multiple repair path-
ways that act at different steps, in-
cluding specialized endonucleases that
incise the lesion after replication fork
arrest, homologous recombination pro-
teins that repair the resultant DSBs,
and translesion polymerases that repli-
cate past the damaged base (Mirchan-
dani and D’Andrea, 2006; Wang and
D’Andrea, 2004) (Figure 1). A major func-
tion of the FA proteins appears to be to
coordinate each of these three indepen-
dent repair pathways (Knipscheer et al.,
2009; Mirchandani and D’Andrea, 2006).
In addition to a direct role in promoting
efficient ICL repair, it was predicted
several years ago that FA proteins might
Molecular Cell 39, July 30, 2010 ª2010 Elsevier Inc.