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Human DNA polymerase ι (Polι) is one of the Y-family DNA polymerases involved in translesion synthesis (TLS), which allows continued replication at damaged DNA templates. Polι has a noncanonical PCNA-interacting protein box (PIP-box) within an internal region of the protein. Polι activity is stimulated by PCNA binding through the noncanonical PIP-box. To clarify the interaction of PCNA with the noncanonical PIP-box of Polι, PCNA and a Polι peptide carrying the noncanonical PIP-box complex have been cocrystallized. The crystal belongs to space group C2, with unit-cell parameters a = 167.1, b = 68.7, c = 90.0 Å, β = 95.1°. Structural analysis by molecular replacement is in progress.
Genomic DNA is constantly damaged by endogenous and environmental factors. DNA damage in the S-phase often stalls DNA replication by the replicative polymerases Polδ or Pol because they possess compact active sites. Translesion synthesis (TLS) is one of the DNA damage-tolerance mechanisms which allows the continuation of DNA synthesis at damaged templates. During TLS, Polδ or Pol are replaced by specialized DNA polymerases which can perform DNA synthesis using damaged templates. The specialized polymerases are termed ‘TLS polymerases’ and include Y-family DNA polymerases such as Polη, Polκ and Polι (Ohmori et al., 2001 ). Because of their wide active sites, the three TLS polymerases mentioned above can accommodate DNA templates containing bulky damaged bases. Thus, TLS polymerases can perform bypass synthesis using damaged DNA templates in a lesion-specific manner. For instance, Polη is able to efficiently incorporate two adenines opposite a thymine–thymine (T–T) cyclobutane pyrimidine dimer (Masutani et al., 2000 ), which is one of the major photoproducts resulting from UV irradiation. Polι efficiently incorporates an adenine opposite the 3′-T of the 6–4 T–T pyrimidine–pyrimidone photoproduct (Tissier et al., 2000 ; Vaisman et al., 2003 ). Polκ bypasses dG-N2-BPDE (benzo[a]pyrene diol epoxide) by inserting the correct cytosine opposite the bulky lesion (Suzuki et al., 2002 ; Zhang et al., 2000 , 2002 ).
Proliferating cell nuclear antigen (PCNA) forms a ring-shaped homotrimer with a molecular weight of ~90 kDa and functions as a sliding clamp which tethers DNA polymerases onto primer termini. Many proteins involved in replication, repair, the cell cycle, chromatin assembly and sister-chromatid cohesion interact with PCNA (Moldovan et al., 2007 ). Most such proteins have a PCNA-interacting protein box (termed a ‘PIP-box’; Warbrick, 1998 ). The canonical PIP-box is composed of eight amino-acid residues Qxxhxxaa, where the first residue Q is conserved, h is a hydrophobic residue such as Met, Leu or Ile and a is an aromatic residue such as Phe or Tyr. To date, the structures of human PCNA bound to peptides derived from human p21, the p66 subunit of human Polδ, an artificial PL peptide and the full-length human flap endonuclease I protein have been determined (Gulbis et al., 1996 ; Bruning & Shamoo, 2004 ; Kontopidis et al., 2005 ; Sakurai et al., 2005 ). The peptides and protein all include a canonical PIP-box located at the extreme C-terminus and the structures revealed that the interactions of the canonical PIP-boxes with PCNA are very similar to each other. In contrast, the PIP-boxes of the abovementioned three TLS polymerases do not contain the canonical sequence: they lack the conserved Gln residue. The canonical Gln residue is replaced by Met, Lys and Lys in Polη, Polκ and Polι, respectively, and it has been reported that DNA synthesis by these TLS polymerases is stimulated by PCNA binding via their noncanonical PIP-boxes (Haracska et al., 2001a ,b ; Haracska et al., 2002 ; Vidal et al., 2004 ).
Human Polι is composed of 715 amino acids and has a molecular weight of 80 kDa (McDonald et al., 1999 ). The noncanonical PIP-box of Polι has been identified as 420-KKGLIDYY-427 (Vidal et al., 2004 ) and is located in an internal region of the protein, whereas Polη and Polκ have noncanonical PIP-boxes at their extreme C-termini. To reveal the interaction of human PCNA with the internal noncanonical PIP-box of Polι, we performed a structural study of the PCNA–Polι peptide complex. Here, we report a crystallization and X-ray diffraction study of human PCNA in complex with a Polι peptide carrying the noncanonical PIP-box.
Recombinant PCNA was expressed in Escherichia coli BL21 (DE3) harbouring a pT7-PCNA expression vector (Fukuda et al., 1995 ). To date, various procedures for the preparation of recombinant hPCNA have been reported (Gulbis et al., 1996 ; Zheleva et al., 2000 ; Sakurai et al., 2005 ; Bruning & Shamoo, 2004 ; Kontopidis et al., 2005 ). However, the protein was purified in a similar manner to that described previously for the PCNA G178S mutant (Hishiki et al., 2008 ). In brief, bacterial cells were lysed by sonication and the supernatant of the cell lysate was purified using HiTrap Q HP and HiTrap Phenyl HP columns (GE Healthcare). The fractions containing PCNA were finally purified using a HiLoad 26/60 Superdex 200 column (GE Healthcare) equilibrated with 10 mM HEPES–NaOH pH 7.4 and 100 mM NaCl. The purified protein was concentrated to 10 mg ml−1 using Amicon Ultra (Millipore) and frozen using liquid N2.
A peptide composed of residues 415–437 of human Polι was commercially synthesized (GenScript Co., USA). A tenfold molar excess of the Polι peptide (ALNTAKKGLIDYYLMPSLSTTSR) was incubated with PCNA (7.0 mg ml−1). Cocrystals of PCNA–Polι peptide were grown by hanging-drop vapour diffusion at 293 K. Typical hanging drops were prepared by mixing 0.9 µl protein solution and 0.9 µl reservoir solution containing 25% PEG 2K MME, 100 mM sodium cacodylate pH 6.4 and 200 mM potassium thiocyanate. Rod-shaped crystals were obtained in a few weeks (Fig. 1 ). Crystals were transferred to a cryo-buffer (22.5% PEG 3350, 100 mM sodium cacodylate pH 6.4, 200 mM potassium thiocyanate and 20% ethylene glycol) for cryoprotection. Crystals were then flash-frozen in an N2-gas stream at 100 K. X-ray diffraction data were collected from PCNA–Polι peptide crystals using an ADSC Quantum 210 CCD detector on beamline NW12A at Photon Factory Advanced Ring (PF-AR; Fig. 2 ). The diffraction data were indexed, integrated and scaled using HKL-2000 (Otwinowski & Minor, 1997 ). Diffraction data statistics are given in Table 1 . The crystal belongs to space group C2, with unit-cell parameters a = 167.1, b = 68.7, c = 90.0 Å, β = 95.1°. The asymmetric unit is estimated to contain one trimer of PCNA–Polι peptide, with a corresponding crystal volume per protein weight (V M) of 3.0 Å3 Da−1 and a solvent content of 59%. Structural determination by molecular replacement is now in progress.
We acknowledge the kind support of Drs N. Shimizu, K. Hasegawa, H. Sakai, M. Kawamoto and M. Yamamoto during data collection at SPring-8 and Drs Y. Yamada, N. Matsugaki and N. Igarashi and Professor S. Wakatsuki during data collection at Photon Factory. This work was supported by a Grant-in-Aid for Young Scientists (B) from Japan Society for the Promotion of Science (JSPS) to HH (16770080 and 18770091), a Grant-in-Aid for Scientific Research Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to HH (16048226, 17048023 and 19036025) to TS (17054035) and to MS (18054026 and 20051020) and the National Project on Protein Structural and Functional Analyses (Protein 3000 Project) and the Target Protein Research Program to MS, TS and HH from MEXT. We thank Dr Cyrus Vaziri, Department of Pathology and Laboratory Medicine, Boston University School of Medicine for English corrections and discussion.