Repair Scaffolding Reaches New Heights at Blocked Replication Forks

Molecular Cell

Previews in a HSP90ATPgS/AGO1/siRNA duplex complex. In contrast, completing RISC assembly in the presence of ATP showed that AGO1 was mostly loaded with singlestranded siRNA. Based on these observations, Iki and colleagues suggested the following model for plant HSP90-faciliated RISC loading. HSP90 binds to AGO1, 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 dissociation of the chaperone and AGO1, followed by an additional conformational rearrangement in AGO1 that could facilitate 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 before, not after, RISC loading with duplex siRNA. Presumably, this requirement reflects an HSP90-dependent conformational 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 assembly pathways. 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 modulated by specific cochaperones that are needed for HSP90 action during RISC formation? How does the separation of miRNA/miRNA* and siRNA duplexes differ in tobacco AGO1-containing complexes? 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 general.

REFERENCES Carthew, R.W., and Sontheimer, E.J. (2009). Cell 136, 642–655. Iki, T., Yoshikawa, M., Nishikiori, M., Jaudal, M.C., Matsumoto-Yokoyama, E., Mitsuhara, I., Meshi, T., and Ishikawa, M. (2010). Mol. Cell 39, this issue, 282–291. Iwasaki, S., Kobayashi, M., Yoda, M., Sakaguchi, Y., Katsuma, S., Suzuki, T., and Tomari, Y. (2010). Mol. Cell 39, this issue, 292–299. Kawamata, T., and Tomari, Y. (2010). Trends Biochem. Sci. 35, 368–376. Kawamata, T., Seitz, H., and Tomari, Y. (2009). Nat. Struct. Mol. Biol. 16, 953–960. Matranga, C., Tomari, Y., Shin, C., Bartel, D.P., and Zamore, P.D. (2005). Cell 123, 607–620. Nyka¨nen, A., Haley, B., and Zamore, P.D. (2001). Cell 107, 309–321. Tahbaz, N., Carmichael, J.B., and Hobman, T.C. (2001). J. Biol. Chem. 276, 43294–43299. Tahbaz, N., Kolb, F.A., Zhang, H., Jaronczyk, K., Filipowicz, W., and Hobman, T.C. (2004). EMBO Rep. 5, 189–194. Taipale, M., Jarosz, D.F., and Lindquist, S. (2010). Nat. Rev. Mol. Cell Biol. 11, 515–528. Yoda, M., Kawamata, T., Paroo, Z., Ye, X., Iwasaki, S., Liu, Q., and Tomari, Y. (2010). Nat. Struct. Mol. Biol. 17, 17–23.

Repair Scaffolding Reaches New Heights at Blocked Replication Forks Michael Downey,1 Ellen R. Edenberg,1 and David P. Toczyski1,* 1Department of Biochemistry and Biophysics, University of California, San Francisco, 1450 3rd Street, San Francisco, CA 94158-9001, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2010.07.007

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. Early studies of the DNA damage 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 progression, repair or bypass the damage in ques-

tion, and subsequently resume DNA synthesis—all without disrupting the delicate fork structure. This reorganization requires the recruitment of several repair and signaling complexes, in part facilitated by the recruitment of BRCT domain-containing proteins. BRCT do-

162 Molecular Cell 39, July 30, 2010 ª2010 Elsevier Inc.

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

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b11

Dp

Ac thus may serve as scaffolds to and Durocher, 2009). RNF168 Ac coordinate multiple binding recruitment relies on prior Mec1 partners at sites of damage. sumoylation at sites of DNA P P P In this issue, Ohouo et al. damage (Galanty et al., 2009). Pol (2010) use mass spectromTo this end, the Smc5/Smc6 etry to expand the repertoire complex shown to interact 9 Ac tt10 of proteins associated with with Rtt107 in this study inRtt101 R Mms1 Ac these scaffolds in damagecludes the sumo ligase Mms21 7 Smc5/6 M 0 m s22 1 P Hrt1 Ub Rtt Substrate X treated cells. They show that (Ohouo et al., 2010). Identifying Nse5 Kre29 Rtt107 associates with Dpb11, the substrates of Mms21 and Mms21 P as well as the Slx4, Rtt101, Rtt101 will reveal how these Su Substrate Y and Smc5/Smc6 complexes. proteins aid in repair at broken 4 x l S Ohouo et al. (2010) identify replication forks. One question arising from Rtt107 interactors using Slx1 this work is whether the SILAC to quantitatively comRad1-Rad10 pare proteins recovered in Rtt107 scaffold is temporally paired Rtt107 immunoprecipor spatially remodeled during Figure 1. A Model for Scaffolding at a Stalled Replication Fork itates. Specifically, they identhe course of a repair event The BRCT scaffold Rtt107 and the Cul4-related Rtt101 become chromatin tify a Mec1-dependent interand whether its binding partassociated in a mutually dependent manner when replication forks are blocked action between Dpb11 and ners vary with the type of lesion. by damage (blue star). The recruitment of both complexes depends upon the HAT Rtt109, which has established roles in both unperturbed replication and Rtt107 (Figure 1). During unBecause each tandem BRCT upon replication stress. The S. pombe Rtt107 homolog Brc1 has been shown perturbed DNA replication, domain likely interacts with to associate with the Mec1 target H2A(X). Here, the authors show that Mec1 Dpb11 has a separate, esone phosphoepitope at a time, (hATR) phosphorylation of Slx4 promotes an association with the BRCT scaffold Dpb11. This association also requires the presence of the Rtt107 scaffold. sential function in origin firing it is possible that the BRCT Slx4 itself is thought to target many structure-specific nucleases to their sites in which it simultaneously domains of the scaffold seof action. Rtt107 is shown by Ohouo et al. to also be associated with the Smc5/ binds two replication factors quentially bind and exchange Sm6 complex, which contains a sumo ligase. Touching proteins represent through its BRCT domains. different repair proteins. Ohouo binding dependencies, but not necessarily direct interactions. In response to DNA damage, et al. (2010) provide evidence Dpb11 is recruited to stalled that the Slx4-mediated interacreplication forks and is now linked for Despite the fact that the N terminus of tion between Dpb11 and Rtt107 is parthe first time to Rtt107 and Slx4 (Ohouo Rtt107, containing four of its six BRCT ticularly important for resistance to MMS, et al., 2010). Both Rtt107 and Slx4 have domains, is necessary and sufficient for its consistent with the fact that Slx4 mutants previously been implicated in replication interaction with Slx4, this interaction does are particularly sensitive to this damage fork restart following transient exposure not appear to require DNA damage or the agent. In contrast, Rtt107 mutants are sensito DNA-damaging agents (Rouse, 2004; checkpoint pathway (Roberts et al., 2006; tive to a wide array of DNA-damaging Roberts et al., 2006). Slx4 itself is thought Ohouo et al., 2010). This suggests that the agents (Roberts et al., 2008). Whereas the of as a scaffold for multiple structure- interaction is mediated by a kinase that is Slx4/Rtt107 complex binds Dpb11 after specific endonuclease complexes, in- not regulated by damage or that some other treatment with MMS, binding of Rtt107 to cluding Slx1 and Rad1/Rad10 (Fricke domain adjacent to the N-terminal BRCTs chromatin depends on both Rtt101 and and Brill, 2003; Flott et al., 2007). In addi- mediates binding to Slx4. the Rtt109 histone acetyltransferase (Robtion, Slx4 has previously been shown to Important clues to the function of the erts et al., 2008). Perhaps, different combe required for Rtt107 phosphorylation Rtt107 scaffold in replication fork repair plexes are assembled following treatment in response to DNA damage, although come from the observation that it binds with different damaging agents. Slx4’s interthe function of Rtt107 phosphorylation is the Rtt101 and Smc5/Smc6 complexes actions with Rtt107 and the Rad1/Rad10 debated (Rouse, 2004; Roberts et al., (Ohouo et al., 2010; Roberts et al., 2008). nuclease appear to be mutually exclusive, 2006; Ohouo et al., 2010). Here, the The Rtt101 cullin and its binding partners, suggesting that these associations may authors show that Mec1 phosphorylation Mms1 and Mms22, form a Cul4-like ubiqui- also be context dependent (Flott et al., of Slx4 is required for its binding to tin ligase complex whose substrates are 2007). Intriguingly, Brc1, the Rtt107 Dpb11 (Ohouo et al., 2010). This phos- unknown (Zaidi et al., 2008). Rtt101 may homolog in S. pombe, binds to the phorylation is likely to mediate a direct ubiquitinate a protein whose destruction Mec1-dependent gH2AX phosphoepitope interaction with some of Dpb11’s BRCT by the proteasome is required for fork (Williams et al., 2010). Whether this interdomains, although confirmation of this will restart, or it may assemble ubiquitin chains action is conserved in budding yeast await further analysis. In addition, deletion that contribute to existing scaffold struc- remains to be tested. A sampling of the of the gene encoding the downstream tures at sites of damage. In mammalian Rtt107 interactome at different times kinase Rad53 (hChk2) also eliminates cells, RNF8 and RNF168 ubiquitin ligases during the course of repair or following most, but not all, of the Slx4-Dpb11 interac- collaborate to attach ubiquitin chains onto treatment with different damaging agents tion, hinting at more complicated higher- histones at DNA lesions, which recruit will shed important light on the plasticity order interactions (Ohouo et al., 2010). checkpoint and DNA repair proteins (Panier of repair scaffolds.

Molecular Cell 39, July 30, 2010 ª2010 Elsevier Inc. 163

Molecular Cell

Previews Finally, the description of Rtt107, Slx4, and Dpb11 as scaffold proteins that function solely to recruit downstream factors may be an oversimplification. Dpb11 (TopBP1) and Slx4 have demonstrated roles in the direct activation of Mec1 kinase and Slx1 nuclease activities, respectively (Kumagai et al., 2006; Fricke and Brill, 2003). Perhaps there are also additional, catalytic functions of Rtt107 yet to be described.

and Rouse, J. (2007). Mol. Cell. Biol. 27, 6433– 6445. Fricke, W.M., and Brill, S.J. (2003). Genes Dev. 17, 1768–1778. Galanty, Y., Belotserkovskaya, R., Coates, J., Polo, S., Miller, K.M., and Jackson, S.P. (2009). Nature 462, 935–939.

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– 180. Rouse, J. (2004). EMBO J. 23, 1188–1197.

Kumagai, A., Lee, J., Yoo, H.Y., and Dunphy, W.G. (2006). Cell 124, 943–955.

REFERENCES

Ohouo, P.Y., Bastos de Oliveira, F.M., Almeida, B.S., and Smolka, M.B. (2010). Mol. Cell 39, this issue, 300–306.

Flott, S., Alabert, C., Toh, G.W., Toth, R., Sugawara, N., Campbell, D.G., Haber, J.E., Pasero, P.,

Panier, S., and Durocher, D. (2009). DNA Repair (Amst.) 8, 436–443.

Williams, J.S., Williams, R.S., Dovey, C.L., Guenther, G., Tainer, J.A., and Russell, P. (2010). EMBO J. 29, 1136–1148. Zaidi, I.W., Rabut, G., Poveda, A., Scheel, H., Malmstro¨m, J., Ulrich, H., Hofmann, K., Pasero, P., Peter, M., and Luke, B. (2008). EMBO Rep. 9, 1034–1040.

Dangerous Liaisons: Fanconi Anemia and Toxic Nonhomologous End Joining in DNA Crosslink Repair Samuel F. Bunting1 and Andre´ Nussenzweig1,* 1Experimental Immunology Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2010.07.016

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 anemia (FA) pathway is involved in preventing aberrant DNA repair. These studies suggest a potentially significant 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 basis of 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 FANCD2FANCI. Cells from FA patients of all subtypes exhibit chromosome abnormalities when treated with DNA crosslinking agents such as mitomycin C (Moldovan and D’Andrea, 2009; Wang and D’Andrea, 2004), which block replication and transcription. Defective interstrand crosslink (ICL) repair is thought to underlie the clinical and cellular pheno-

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types associated with FA. DNA crosslink processing utilizes multiple repair pathways that act at different steps, including specialized endonucleases that incise the lesion after replication fork arrest, homologous recombination proteins that repair the resultant DSBs, and translesion polymerases that replicate past the damaged base (Mirchandani and D’Andrea, 2006; Wang and D’Andrea, 2004) (Figure 1). A major function of the FA proteins appears to be to coordinate each of these three independent 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