Asami Hishiki

Structural Basis for Novel Interactions between Human Translesion Synthesis Polymerases and Proliferating Cell Nuclear Antigen

Translesion synthesis (TLS) is a DNA damage tolerance mechanism that allows continued DNA synthesis, even in the presence of damaged DNA templates. Mammals have multiple DNA polymerases specialized for TLS, including Polη, Polι, and Polκ. These enzymes show preferential bypass for different lesions. Proliferating cell nuclear antigen (PCNA), which functions as a sliding clamp for the replicative polymerase Polδ, also interacts with the three TLS polymerases. Although many PCNA-binding proteins have a highly conserved sequence termed the PCNA-interacting protein box (PIP-box), Polη, Polι, and Polκ have a noncanonical PIP-box sequence. In response to DNA damage, Lys-164 of PCNA undergoes ubiquitination by the RAD6-RAD18 complex, and the ubiquitination is considered to facilitate TLS. Consistent with this, these three TLS polymerases have one or two ubiquitin binding domains and are recruited to replication forks via interactions with ubiquitinated PCNA involving the noncanonical PIP-box and ubiquitin binding domain. However, it is unclear how these TLS polymerases interact with PCNA. To address the structural basis for interactions between different TLS polymerases and PCNA, we determined crystal structures of PCNA bound to peptides containing the noncanonical PIP-box of these polymerases. We show that the three PIP-box peptides interact with PCNA in different ways, both from one another and from canonical PIP-box peptides. Especially, the PIP-box of Polι adopts a novel structure. Furthermore, these structures enable us to speculate how these TLS polymerases interact with Lys-164-monoubiquitinated PCNA. Our results will provide clues to understanding the mechanism of preferential recruitment of TLS polymerases to the stalled forks.

Identification of small molecule proliferating cell nuclear antigen (PCNA) inhibitor that disrupts interactions with PIP-box proteins and inhibits DNA replication

Background: PCNA is a multifunctional component of DNA replication and repair machinery. Results: A novel small molecule inhibitor of the PCNA protein-protein interaction inhibited DNA replication, induced DNA replication stress, and increased cisplatin-mediated DNA damage response in cells. Conclusion: The biochemical PCNA inhibitor can inhibit PCNA functions essential for cells. Significance: Inhibition of the PCNA protein-protein interaction can be a new strategy to sensitize cancer cells to chemotherapy. We have discovered that 3,3′,5-triiodothyronine (T3) inhibits binding of a PIP-box sequence peptide to proliferating cell nuclear antigen (PCNA) protein by competing for the same binding site, as evidenced by the co-crystal structure of the PCNA-T3 complex at 2.1 Å resolution. Based on this observation, we have designed a novel, non-peptide small molecule PCNA inhibitor, T2 amino alcohol (T2AA), a T3 derivative that lacks thyroid hormone activity. T2AA inhibited interaction of PCNA/PIP-box peptide with an IC50 of ∼1 μm and also PCNA and full-length p21 protein, the tightest PCNA ligand protein known to date. T2AA abolished interaction of PCNA and DNA polymerase δ in cellular chromatin. De novo DNA synthesis was inhibited by T2AA, and the cells were arrested in S-phase. T2AA inhibited growth of cancer cells with induction of early apoptosis. Concurrently, Chk1 and RPA32 in the chromatin are phosphorylated, suggesting that T2AA causes DNA replication stress by stalling DNA replication forks. T2AA significantly inhibited translesion DNA synthesis on a cisplatin-cross-linked template in cells. When cells were treated with a combination of cisplatin and T2AA, a significant increase in phospho(Ser139)histone H2AX induction and cell growth inhibition was observed.

Structure of a Novel DNA-binding Domain of Helicase-like Transcription Factor (HLTF) and Its Functional Implication in DNA Damage Tolerance

Background: HLTF is responsible for template-switching of DNA damage tolerance; HLTF has a novel DNA-binding HIRAN domain, but its function is unknown. Results: The structure of HIRAN domain bound to DNA reveals that the domain recognizes 3′-end of DNA. Conclusion: HLTF is recruited to a damaged site via interaction of the HIRAN domain with 3′-end. Significance: The structure provides a structural basis for the mechanism of template-switching. HLTF (helicase-like transcription factor) is a yeast RAD5 homolog found in mammals. HLTF has E3 ubiquitin ligase and DNA helicase activities, and plays a pivotal role in the template-switching pathway of DNA damage tolerance. HLTF has an N-terminal domain that has been designated the HIRAN (HIP116 and RAD5 N-terminal) domain. The HIRAN domain has been hypothesized to play a role in DNA binding; however, the structural basis of, and functional evidence for, the HIRAN domain in DNA binding has remained unclear. Here we show for the first time the crystal structure of the HIRAN domain of human HLTF in complex with DNA. The HIRAN domain is composed of six β-strands and two α-helices, forming an OB-fold structure frequently found in ssDNA-binding proteins, including in replication factor A (RPA). Interestingly, this study reveals that the HIRAN domain interacts with not only with a single-stranded DNA but also with a duplex DNA. Furthermore, the structure unexpectedly clarifies that the HIRAN domain specifically recognizes the 3′-end of DNA. These results suggest that the HIRAN domain functions as a sensor to the 3′-end of the primer strand at the stalled replication fork and that the domain facilitates fork regression. HLTF is recruited to a damaged site through the HIRAN domain at the stalled replication fork. Furthermore, our results have implications for the mechanism of template switching.

A Small Molecule Inhibitor of Monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA) Inhibits Repair of Interstrand DNA Cross-link, Enhances DNA Double Strand Break, and Sensitizes Cancer Cells to Cisplatin

Background: Lys-164-monoubiquitinated PCNA is essential for interstrand DNA cross-link (ICL) repair. Results: A small molecule, T2AA, bi-molecularly binds to PCNA at a PIP-box cavity and close to Lys-164. T2AA inhibited monoubiquitinated PCNA interactions and ICL repair and enhanced DNA double strand breaks. Conclusion: An inhibitor of monoubiquitinated PCNA inhibits ICL repair. Significance: Inhibition of monoubiquitinated PCNA could improve chemotherapeutic efficacy. Small molecule inhibitors of proliferating cell nuclear antigen (PCNA)/PCNA interacting protein box (PIP-Box) interactions, including T2 amino alcohol (T2AA), inhibit translesion DNA synthesis. The crystal structure of PCNA in complex with T2AA revealed that T2AA bound to the surface adjacent to the subunit interface of the homotrimer of PCNA in addition to the PIP-box binding cavity. Because this site is close to Lys-164, which is monoubiquitinated by RAD18, we postulated that T2AA would affect monoubiquitinated PCNA interactions. Binding of monoubiquitinated PCNA and a purified pol η fragment containing the UBZ and PIP-box was inhibited by T2AA in vitro. T2AA decreased PCNA/pol η and PCNA/REV1 chromatin colocalization but did not inhibit PCNA monoubiquitination, suggesting that T2AA hinders interactions of pol η and REV1 with monoubiquitinated PCNA. Interstrand DNA cross-links (ICLs) are repaired by mechanisms using translesion DNA synthesis that is regulated by monoubiquitinated PCNA. T2AA significantly delayed reactivation of a reporter plasmid containing an ICL. Neutral comet analysis of cells receiving T2AA in addition to cisplatin revealed that T2AA significantly enhanced formation of DNA double strand breaks (DSBs) by cisplatin. T2AA promoted colocalized foci formation of phospho-ATM and 53BP1 and up-regulated phospho-BRCA1 in cisplatin-treated cells, suggesting that T2AA increases DSBs. When cells were treated by cisplatin and T2AA, their clonogenic survival was significantly less than that of those treated by cisplatin only. These findings show that the inhibitors of monoubiquitinated PCNA chemosensitize cells by inhibiting repair of ICLs and DSBs.

En bloc transfer of polyubiquitin chains to PCNA in vitro is mediated by two different human E2–E3 pairs

Post-replication DNA repair in eukaryotes is regulated by ubiquitination of proliferating cell nuclear antigen (PCNA). Monoubiquitination catalyzed by RAD6–RAD18 (an E2–E3 complex) stimulates translesion DNA synthesis, whereas polyubiquitination, promoted by additional factors such as MMS2–UBC13 (a UEV–E2 complex) and HLTF (an E3 ligase), leads to template switching in humans. Here, using an in vitro ubiquitination reaction system reconstituted with purified human proteins, we demonstrated that PCNA is polyubiquitinated predominantly via en bloc transfer of a pre-formed ubiquitin (Ub) chain rather than by extension of the Ub chain on monoubiquitinated PCNA. Our results support a model in which HLTF forms a thiol-linked Ub chain on UBC13 (UBC13∼Ubn) and then transfers the chain to RAD6∼Ub, forming RAD6∼Ubn+1. The resultant Ub chain is subsequently transferred to PCNA by RAD18. Thus, template switching may be promoted under certain circumstances in which both RAD18 and HLTF are coordinately recruited to sites of stalled replication.

Crystal structure of zinc-finger domain of Nanos and its functional implications

Nanos is an RNA‐binding protein that is involved in the development and maintenance of germ cells. In combination with Pumilio, Nanos binds to the 3′ untranslated region of a messenger RNA and represses its translation. Nanos has two conserved Cys‐Cys‐His‐Cys zinc‐finger motifs that are indispensable for its function. In this study, we have determined the crystal structure of the zinc‐finger domain of zebrafish Nanos, for the first time revealing that Nanos adopts a novel zinc‐finger structure. In addition, Nanos has a conserved basic surface that is directly involved in RNA binding. Our results provide the structural basis for further studies to clarify Nanos function.

Lipid Nanoparticle-mediated siRNA Transfer Against PCTAIRE1/PCTK1/Cdk16 Inhibits In Vivo Cancer Growth.

PCTAIRE1/CDK16/PCTK1 plays critical roles in cancer cell proliferation and antiapoptosis. To advance our previously published in vitro results with PCTAIRE1 silencing, we examined the in vivo therapeutic potential of this approach by using small interfering RNA (siRNA) encapsulated by lipid nanoparticles. Therapy experiments of PCTAIRE1 siRNA were performed using human HCT116 colorectal cancer cells and human A2058 melanoma cells. A single dose of PCTAIRE1 siRNA-lipid nanoparticles was found to be highly effective in reducing in vivo PCTAIRE1 expression for up to 4 days as assayed by immunoblotting. Therapy experiments were started 4 days after subcutaneous injection of cancer cells. Treatment with PCTAIRE1 siRNA-lipid nanoparticles (0.5 mg/kg RNA, twice a week) reduced tumor volume and weight significantly compared with the scramble-control group. Histopathological analysis (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) showed increased apoptosis of tumor cells treated with PCTAIRE1-siRNA. Overall, our results demonstrate that siRNA treatment targeting PCTAIRE1 is effective in vivo, suggesting that PCTAIRE1 siRNA-lipid nanoparticles might be a novel therapeutic approach against cancer cells.PCTAIRE1/CDK16/PCTK1 plays critical roles in cancer cell proliferation and antiapoptosis. To advance our previously published in vitro results with PCTAIRE1 silencing, we examined the in vivo therapeutic potential of this approach by using small interfering RNA (siRNA) encapsulated by lipid nanoparticles. Therapy experiments of PCTAIRE1 siRNA were performed using human HCT116 colorectal cancer cells and human A2058 melanoma cells. A single dose of PCTAIRE1 siRNA-lipid nanoparticles was found to be highly effective in reducing in vivo PCTAIRE1 expression for up to 4 days as assayed by immunoblotting. Therapy experiments were started 4 days after subcutaneous injection of cancer cells. Treatment with PCTAIRE1 siRNA-lipid nanoparticles (0.5 mg/kg RNA, twice a week) reduced tumor volume and weight significantly compared with the scramble-control group. Histopathological analysis (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) showed increased apoptosis of tumor cells treated with PCTAIRE1-siRNA. Overall, our results demonstrate that siRNA treatment targeting PCTAIRE1 is effective in vivo, suggesting that PCTAIRE1 siRNA-lipid nanoparticles might be a novel therapeutic approach against cancer cells.

Crystallographic study of G178S mutant of human proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) is an evolutionarily conserved protein that forms a ring-shaped homotrimer that functions as a sliding clamp for DNA replication. The rev6-1 mutation of Saccharomyces cerevisiae, which inactivates both translesion DNA synthesis and damage-avoidance pathways while having little effect on normal cell growth, has a G178S substitution in the PCNA protein. Human PCNA protein carrying the G178S substitution was crystallized. Two crystal forms were obtained under similar conditions. Crystal forms I and II belong to space groups P2(1), with unit-cell parameters a = 84.1, b = 130.2, c = 97.8 A, beta = 113.4 degrees , and P2(1)2(1)2(1), with unit-cell parameters a = 68.1, b = 100.2, c = 131.2 A, respectively. Structural analyses by molecular replacement are now in progress.

Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins.

Crystallographic study of a novel DNA-binding domain of human HLTF involved in the template-switching pathway to avoid the replication arrest caused by DNA damage.

HLTF is a pivotal protein in the template-switching pathway that allows DNA synthesis to continue even in the presence of DNA damage by utilizing a newly synthesized undamaged strand as a template. HLTF has a novel DNA-binding domain termed HIRAN that has been recently found in various proteins, although its detailed function remains unclear. In this study, the HIRAN domain of human HLTF was successfully crystallized. The crystals belonged to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 130.0, c = 150.1 Å.