Replication Protein A-Mediated Recruitment and Activation of Rad17 Complexes

Replication protein A-mediated recruitment and activation of Rad17 complexes Lee Zou*†‡§, Dou Liu†, and Stephen J. Elledge*†‡§¶ʈ *Brigham and Women’s Hospital, †Howard Hughes Medical Institute, and ‡Department of Genetics, Harvard Medical School, Boston, MA 02115; and §Verna and Marrs McLean Department of Biochemistry and Molecular Biology and ¶Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030 Contributed by Stephen J. Elledge, September 23, 2003 The human Rad17–Rfc2-5 and Rad9–Rad1–Hus1 complexes play structures used by RFC to load PCNA. The Rad24 complex, the crucial roles in the activation of the ATR-mediated DNA damage budding yeast counterpart of the human Rad17 complex, was and DNA replication stress response pathways. In response to DNA also shown to recruit the PCNA-like Ddc1–Mec3–Rad17 com- damage, Rad9 is recruited to chromatin in a Rad17-dependent plex onto gapped plasmids (12). Although these studies provided manner in human cells. However, the DNA structures recognized evidence that the Rad17 complex could bring the Rad9 complex by the Rad17–Rfc2-5 complex during the damage response have to DNA in vitro, they did not reveal any difference between the not been defined. Here, we show that replication protein A (RPA) DNA structure specificities of the Rad17 complex and RFC. stimulates the binding of the Rad17–Rfc2-5 complex to single- Why the function of the Rad17 complex is regulated by DNA stranded DNA (ssDNA), primed ssDNA, and a gapped DNA struc- damage remains unanswered. ture. Furthermore, RPA facilitates the recruitment of the Rad9– RPA-coated ssDNA is a common structure generated at the Rad1–Hus1 complex by the Rad17–Rfc2-5 complex to primed and sites of DNA damage, and it plays an important role in the gapped DNA structures in vitro. These findings suggest that RPA- recruitment of the ATR–ATRIP complex to DNA lesions (3). coated ssDNA is an important part of the structures recognized by RPA is known to facilitate the specific binding of RFC to the the Rad17–Rfc2-5 complex. Unlike replication factor C (RFC), which primer͞template junction, and it stimulates the loading of PCNA uses the 3؅ primer͞template junction to recruit proliferating cell by RFC in vitro (13). Whether RPA has a role in regulating the nuclear antigen (PCNA), the Rad17–Rfc2-5 complex can use both activity and͞or DNA-structure specificity of the Rad17 complex the 5؅ and the 3؅ primer͞template junctions to recruit the Rad9– is not known. In budding yeast, rfc4-2, a mutant allele of the Rad1–Hus1 complex, and it shows a preference for gapped DNA RFC4 gene, is defective for the checkpoint responses and structures. These results explain how the Rad17–Rfc2-5 complex sensitive to hydroxyurea (HU)-induced replication blocks (14). senses DNA damage and DNA replication stress to initiate check- Interestingly, the HU sensitivity of the rfc4-2 mutant can be point signaling. suppressed by specific mutant alleles of the RFA1 gene (14), suggesting that RPA and the RFC-like checkpoint complex he maintenance of genomic stability requires cells to dupli- might function in concert during the checkpoint response. Tcate and segregate their genomes precisely and coordinately. In this study, we found that the yeast rfa1-t11 mutant is In response to DNA damage or replication stress, cells activate defective for the recruitment of Ddc1 to DNA damage in vivo. a complex signaling pathway to halt genome duplication and In vitro, human RPA stimulates the loading of the Rad17 segregation, to stabilize the replication forks that encounter complex onto linear ssDNA, primed templates and gapped interference, and to promote DNA repair or apoptosis (1). Two templates. Furthermore, RPA stimulates the ability of Rad17 to large protein kinases, ATM and ATR, are the central compo- recruit the Rad9 complex to primed or gapped templates. Unlike nents of the damage response pathway. Whereas ATM primarily RFC, the Rad17 complex can use both the 5Ј and the 3Ј responds to double-stranded DNA breaks (DSB), ATR is im- primer͞template junctions to recruit the Rad9 complex, and it portant for the responses to DSB as well as a variety of types of exhibits a clear preference to the DNA templates with single- DNA damage that interfere with DNA replication (2). We stranded gaps. The unique DNA-structure specificity of the recently showed that ATRIP, the regulatory partner of ATR, Rad17 complex might explain how it recognizes various types of specifically recognizes RPA-coated single-stranded DNA DNA damage in vivo. These findings suggest that RPA is not only (ssDNA) and thereby localizes the ATR–ATRIP complex to the important for the recruitment of the ATR–ATRIP complex to sites of DNA damage (3). Independently of the ATR–ATRIP DNA damage, but is also critical for damage recognition by the complex, the Rad17–Rfc2-5 and the Rad9–Rad1–Hus1 com- Rad17 complex. plexes (referred below as the Rad17 and the Rad9 complexes, respectively), two protein complexes required for the ATR- Materials and Methods mediated response, may also function in the sensing of DNA Yeast Strains. The yeast strain carrying wild-type RFA1, DDC1- damage (4–6). However, how these complexes recognize DNA 2HA, Gal-HO, and a single HO cleavage site (Y2326) was damage and how they interact with ATR–ATRIP on DNA have derived from yJK8-1 provided by D. Toczyski (University of not been biochemically elucidated. California at San Francisco). To generate the rfa1-t11 mutant in The Rad17 complex is a replication factor C (RFC)-like this strain background, the promoter and a 5Ј portion of the protein complex in which Rfc1 is substituted by Rad17, a protein RFA1 gene was cloned into pRS406, and the rfa1-t11 mutation BIOCHEMISTRY homologous to all five subunits of RFC (7, 8). The Rad9 complex (G104A) was introduced by site-directed mutagenesis. The is a ring-shape protein complex that resembles PCNA (9). During DNA replication, RFC specifically recognizes the 3Ј- primer͞template junction and enables PCNA, the sliding clamp Abbreviations: DSB, double-stranded DNA breaks; ssDNA, single-stranded DNA; RFC, replication factor C; PCNA, proliferating cell nuclear antigen. of DNA polymerases, to encircle DNA (10). Analogously, Rad17 ʈTo whom correspondence should be addressed at: Department of Genetics, Center for is required for the recruitment of Rad9 complexes to DNA Genetics and Genomics, Howard Hughes Medical Institute, Room 158D, New Research damage in vivo (4–6). A recent biochemical study (11) reported Building, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail: that the recombinant human Rad17 complex could recruit the [email protected]. Rad9 complex to nicked or gapped plasmids, the same DNA © 2003 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.2336100100 PNAS ͉ November 25, 2003 ͉ vol. 100 ͉ no. 24 ͉ 13827–13832 Downloaded by guest on September 26, 2021 plasmid carrying the rfa1-t11 mutation was then linearized with SpeI and transformed into Y2326 cells. The rfa1-t11 mutation in the resultant yeast strain (Y2327) was verified by sequencing the PCR products derived from the RFA1 locus. Expression and Purification of the Protein Complexes. The expression and purification of the Rad17 and Rad9 complexes were performed essentially as described (8). The human RPA was expressed in Escherichia coli and purified as described in ref. 15. DNA Templates. To generate the DNA templates used in the DNA-binding assays, Ϸ5 pmols of single-stranded pCR-Script DNA was incubated with 25 pmols of various biotinylated DNA oligomers in 100 ␮l of annealing buffer (10 mM Tris⅐HCl, pH 7.5͞100 mM NaCl). The mixture of circular ssDNA and oligomers was heated at 95°C for 3 min and cooled down slowly. The unannealed oligomers were then removed with Qiagen (Valencia, CA) PCR purification columns. In the experiments using preattached DNA templates, the DNA templates were incubated with Dynal (Great Neck, NY) beads coated with streptavidin for 30 min. After the incubation, the beads were washed twice with annealing buffer to remove unattached DNA. DNA-Binding Assays for the Rad17 Complex. Approximately 0.5 pmol of ssDNA or primed ssDNA, 0.5 pmol of the purified Rad17 complex, and various amounts of RPA were incubated in Fig. 1. Defective recruitment of Ddc1 to DNA damage in the rfa1-t11 ␮ ⅐ ͞ mutant. (A) rfa1-t11 mutant cells carrying DDC1-GFP, Gal-HO, and a single HO 500 l of binding buffer (40 mM Tris HCl, pH 7.5 150 mM ͞ ͞ ͞ ␮ ͞ ͞ ͞ cleavage site, and the isogenic RFA1 cells were synchronized at G2 M with NaCl 10 mM MgCl2 100 g/ml BSA 1mMDTT 1mMATP nocodazole for3hat30°C. The cells were then cultured in the presence or 10% glycerol) at room temperature for 30 min. Subsequently, absence of 2% galactose for another 4 h and were analyzed by chromatin streptavidin-coated Dynal beads were added to the reactions, immunoprecipitation with antibodies to GFP. HO, PCR products derived from and incubations were continued for another 30 min. After the a locus 0.5 kb from the HO-induced break. Tub, PCR products derived from a incubation, the beads were retrieved and washed three times control locus at TUB1.(B) Quantification of the Ddc1 signal at the HO locus with binding buffer containing 250 mM NaCl and 0.25 mM ATP. relative to the TUB1 locus from the samples shown in A. Finally, the proteins associated with the beads were released by boiling in denaturing sample buffer and analyzed by SDS͞PAGE and immunoblotting. human Rad17, but is independent of Mec1, the yeast homologue of ATR (4, 5).

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