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Structure and Functional Implications of the Human Rad9-Hus1-Rad1

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Structure and Functional Implications of the Human Rad9-Hus1-Rad1 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/06/17/C109.022384.DC1.html ACCELERATED PUBLICATION This paper is available online at www.jbc.org THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 31, pp. 20457–20461, July 31, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Structure and Functional onto DNA lesion sites by Rad17-RFC2–5 (which consists of one large subunit, Rad17, and four small subunits, RFC2–5), where Implications of the Human it triggers the activation of the cell cycle checkpoint (3, 4). Rad9-Hus1-Rad1 Cell Cycle Moreover, the 9-1-1 complex can also directly participate in DNA repair via physical association with many factors involved *□S Checkpoint Complex in base excision repair (BER), translesion synthesis, homolo- Received for publication, May 18, 2009, and in revised form, June 5, 2009 gous recombination, and mismatch repair pathways (5–9). Published, JBC Papers in Press, June 17, 2009, DOI 10.1074/jbc.C109.022384 Although both the 9-1-1 and the PCNA complexes perform Min Xu‡§, Lin Bai‡§, Yong Gong‡, Wei Xie‡§, Haiying Hang‡1, ‡2 critical functions in eukaryotic cells with predicted similar and Tao Jiang structures (10), their specific roles are distinct. First, the 9-1-1 ‡ From the National Laboratory of Biomacromolecules, Institute of complex is a DNA damage sensor in the cell cycle checkpoint Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 and the §Graduate University of Chinese Academy but does not function as a scaffold for the major DNA replica- of Sciences, 19A Yuquan Road, Shijingshan District, Beijing 100039, China tion factors; however, PCNA plays exactly the opposite role (1, 11). Second, although both the complexes function in DNA Cellular DNA lesions are efficiently countered by DNA repair repair, their specific activities are different. Previous observa- Downloaded from in conjunction with delays in cell cycle progression. Previous tions indicated that some BER enzymes, such as MYH (MutY studies have demonstrated that Rad9, Hus1, and Rad1 can form glycosylate homolog) (12), TDG (thymine DNA glycosylate) a heterotrimeric complex (the 9-1-1 complex) that plays dual (7), and NEIL (Nei-like glycosylate) (8), interact with the 9-1-1 roles in cell cycle checkpoint activation and DNA repair in complex via motifs that are located outside the conserved eukaryotic cells. Although the 9-1-1 complex has been proposed PCNA-interacting box (the PIP box), implying that the 9-1-1 to form a toroidal structure similar to proliferating cell nuclear complex functions as a damage repair-specific clamp, in con- www.jbc.org antigen (PCNA), which plays essential roles in DNA replication trast to PCNA. However, the structural basis for this hypothesis and repair, the structural basis by which it performs different remains unclear. Another important unresolved issue concerns functions has not been elucidated. Here we report the crystal the damage-sensing mechanism of the 9-1-1 complex. During at unknown institution, on October 14, 2009 structure of the human 9-1-1 complex at 3.2 A˚ resolution. The the DNA replication process, the PCNA⅐RFC clamp⅐clamp crystal structure, together with biochemical assays, reveals that loader specifically recognizes the primer-template junction the interdomain connecting loops (IDC loop) of hRad9, hHus1, (13). However, the molecular basis by which the 9-1- and hRad1 are largely divergent, and further cocrystallization 1⅐Rad17-RFC2–5 clamp⅐clamp loader specifically recognizes study indicates that a PCNA-interacting box (PIP box)-contain- the damaged DNA is little known. To address these ques- ing peptide derived from hFen1 binds tightly to the interdomain tions, we performed structural and biochemical studies on connecting loop of hRad1, providing the molecular basis for the the 9-1-1 complex. damage repair-specific activity of the 9-1-1 complex in contrast to PCNA. Furthermore, structural comparison with PCNA EXPERIMENTAL PROCEDURES reveals other unique structural features of the 9-1-1 complex Protein Preparation—The hRad9, hHus1, and hRad1 pro- that are proposed to contribute to DNA damage recognition. teins were co-expressed in a Pichia pastoris expression system. To confirm that these three proteins interact with each other, only hHus1 was constructed with an N-terminal His6 tag, Cellular DNA damage triggers the activation of the cell cycle whereas the other two were constructed without the tag. The checkpoint, leading to a delay or arrest in cell cycle progression full length of His-tagged hHus1 sequence (residues 1–280) was to prevent replication and inducing DNA damage repair (1, 2). cloned into a pPICZ C vector. A full-length hRad1 sequence In response to DNA damage, the 9-1-13 complex can be loaded (residues 1–282) and a truncated hRad9 sequence (residues 1–270) were separately cloned into PmeI-mutated pPICZ C * This research was financially supported by the National Key Basic Research vectors. Two expression cassettes separately containing the Program (Grant 2006CB910902), the National Natural Science Foundation hRad1 gene and the hRad9 gene were then excised from these of China (Grants 30770435, 30721003, and 30530180), the National vectors using the BglII and BamHI sites. The cassettes were High Technology Research and Development Program of China (Grant 2006AA02A319), and the National Natural Science Foundation of China then sequentially ligated into the hHus1-pPICZ C vector, (Grant 30530180). which was linearized with BamHI. The vector, containing The atomic coordinates and structure factors (code 3GGR) have been deposited hRad9, hHus1, and hRad1 genes, was then linearized with in the Protein Data Bank, Research Collaboratory for Structural Bioinformat- ics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). PmeI, transformed, and expressed in the GS115 strain of □S The on-line version of this article (available at http://www.jbc.org) contains P. pastoris to produce the human 9-1-1 heterotrimeric com- supplemental Figs. S1–S7 and supplemental Tables S1 and S2. plex. The expressed complex was purified to homogeneity 1 To whom correspondence may be addressed. Tel.: 86-10-64888473; Fax: via nickel affinity chromatography, Sepharose Q chromatog- 86-10-64888473; E-mail: [email protected]. 2 To whom correspondence may be addressed. Tel.: 86-10-64888510; Fax: 86-10-64888510; E-mail: [email protected]. 3 The abbreviations used are: 9-1-1, Rad9-Hus1-Rad1; PCNA, proliferating cell factor C; hFen1, flap endonuclease 1; PIP box, PCNA-interacting box; BER, nuclear antigen; IDC loop, interdomain connecting loop; RFC, replication base excision repair; GST, glutathione S-transferase. JULY 31, 2009•VOLUME 284•NUMBER 31 JOURNAL OF BIOLOGICAL CHEMISTRY 20457 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/06/17/C109.022384.DC1.html ACCELERATED PUBLICATION: Human Rad9-Hus1-Rad1 Complex raphy, and SuperdexTM200 HR 10/30 gel filtration, and con- Biacore Analysis—Real-time binding and kinetic analyses by centrated to a final concentration of 7 mg/ml in 20 mM Tris- surface plasmon resonance were carried out on a Biacore 3000 HCl, 150 mM NaCl, 1 mM dithiothreitol, and 1 mM EDTA, pH instrument (GE Healthcare, Uppsala, Sweden). The eluent 8.0, for crystallization. contained phosphate-buffered saline and 0.005% Tween 20. Crystal Growth—Crystals were grown at 16 °C by the hang- GST-hRad9 (residues 1–391), GST-hHus1 (residues 1–280), ing drop method, using equal volumes of protein and crystalli- GST-hRad1 (residues 1–282), and GST (as control) were zation solution over a 1-ml reservoir, which was composed of immobilized separately on CM5 chips using an amine coupling 8% polyethylene glycol 2000 monomethyl ether, 100 mM Tris- kit, and the remaining coupling sites were blocked with 1 M HCl (pH 8.0), and 200 mM trimethylamine N-oxide dihydrate. ethanolamine (pH 8.5). Three binding assays were carried out The final plate-shaped single crystals were obtained by micro- using 20 ␮M hFen1 peptide (residues 335–364), 20 ␮M p12 pep- seeding (14). Mercurated crystals were obtained by adding a tide (residues 4–11), and 3 ␮M His-tagged truncated hRad17 final concentration of 1.5 mM Thimerosal (ethylmercuricthio- (residues 78–337), respectively, with 1 mM ATP at 25 °C. All salicylic acid, sodium salt) in the hanging drop and soaking the data collected were analyzed with BIAevaluation software ver- crystals for about 30 h. The diffraction of the crystals was sig- sion 4.1, and the data were fitted to a 1:1 binding model to nificantly improved to 3.2 Å by controlled crystal dehydration obtain equilibrium constants. (15). The crystals were then flash-frozen in the liquid nitrogen before being taken to synchrotron radiation sources for data RESULTS AND DISCUSSION collection. The crystal structure of the human 9-1-1 complex is a closed Downloaded from Data Collection and Structure Determination—3.2 Å native circular ring, with the C-terminal domain of one protein inter- data were collected at Spring8 in Japan, on beamline BL41XU, acting with the N-terminal domain of the following protein in and processed with HKL2000 (16). 3.2 Å mercury derivative the order hRad9 3 hHus1 3 hRad1 (Fig. 1B). Each molecule in data were collected at SLS (Swiss Light Source) in Switzerland, the 9-1-1 complex consists of 18 ␤ sheets (␤I1 to ␤I9 and ␤II1 to ␤ ␣ ␣ ␣ ␣ ␣ on beamline X06SA, and processed with MOSFLM (17). The II9) and four helices ( I1, I2, II1, II2) and is composed of www.jbc.org crystal has space group P21 with one complex in the asymmet- two domains (the N- and C-terminal domains), which are con- ric unit. The phase is determined by single anomalous disper- nected by an interdomain connecting (IDC) loop (Fig.
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