Mutations at multiple CDK phosphorylation consensus sites on Cdt2 increase the affinity of CRL4Cdt2 for PCNA and its ubiquitination activity in S phase.

CRL4Cdt2 ubiquitin ligase plays an important role maintaining genome integrity during the cell cycle. A recent report suggested that Cdk1 negatively regulates CRL4Cdt2 activity through phosphorylation of its receptor, Cdt2, but the involvement of phosphorylation remains unclear. To address this, we mutated all CDK consensus phosphorylation sites located in the C-terminal half region of Cdt2 (Cdt2-18A) and examined the effect on substrate degradation. We show that both cyclinA/Cdk2 and cyclinB/Cdk1 phosphorylated Cdt2 in vitro and that phosphorylation was reduced by the 18A mutation both in vitro and in vivo. The 18A mutation increased the affinity of Cdt2 to PCNA, and a high amount of Cdt2-18A was colocalized with PCNA foci during S phase in comparison with Cdt2-WT. Poly-ubiquitination activity to Cdt1 was concomitantly enhanced in cells expressing Cdt2-18A. Other CRL4Cdt2 substrates, Set8 and thymine DNA glycosylase, begin to accumulate around late S phase to G2 phase, but the accumulation was prevented in Cdt2-18A cells. Furthermore, mitotic degradation of Cdt1 after UV irradiation was induced in these cells. Our results suggest that CDK-mediated phosphorylation of Cdt2 inactivates its ubiquitin ligase activity by reducing its affinity to PCNA, an important strategy for regulating the levels of key proteins in the cell cycle.

Thymine DNA glycosylase modulates DNA damage response and gene expression by base excision repair-dependent and independent mechanisms.

Thymine DNA glycosylase (TDG) is a base excision repair (BER) enzyme, which is implicated in correction of deamination-induced DNA mismatches, the DNA demethylation process and regulation of gene expression. Because of these pivotal roles associated, it is crucial to elucidate how the TDG functions are appropriately regulated in vivo. Here, we present evidence that the TDG protein undergoes degradation upon various types of DNA damage, including ultraviolet light (UV). The UV-induced degradation of TDG was dependent on proficiency in nucleotide excision repair and on CRL4CDT2 -mediated ubiquitination that requires a physical interaction between TDG and DNA polymerase clamp PCNA. Using the Tdg-deficient mouse embryonic fibroblasts, we found that ectopic expression of TDG compromised cellular survival after UV irradiation and repair of UV-induced DNA lesions. These negative effects on cellular UV responses were alleviated by introducing mutations in TDG that impaired its BER function. The expression of TDG induced a large-scale alteration in the gene expression profile independently of its DNA glycosylase activity, whereas a subset of genes was affected by the catalytic activity of TDG. Our results indicate the presence of BER-dependent and BER-independent functions of TDG, which are involved in regulation of cellular DNA damage responses and gene expression patterns.

Cell cycle-dependent subcellular translocation of the human DNA licensing inhibitor geminin.

Once per cell cycle replication is crucial for maintaining genome integrity. Geminin interacts with the licensing factor Cdt1 to prevent untimely replication and is controlled by APC/C-dependent cell cycle specific proteolysis during mitosis and in G1. We show here that human geminin, when expressed in human cells in culture under a constitutive promoter, is excluded from the nucleus during part of the G1 phase and at the transition from G0 to G1. The N-terminal 30 amino acids of geminin, which contain its destruction box, are essential for nuclear exclusion. In addition, 30 amino acids within the central domain of geminin are required for both nuclear exclusion and nuclear accumulation. Cdt1 overexpression targets geminin to the nucleus, while reducing Cdt1 levels by RNAi leads to the appearance of endogenous geminin in the cytoplasm. Our data propose a novel means of regulating the balance of Cdt1/geminin in human cells, at the level of the subcellular localization of geminin.

Alternative replication factor C protein, Elg1, maintains chromosome stability by regulating PCNA levels on chromatin.

Proliferating cell nuclear antigen (PCNA) is loaded on chromatin upon initiation of the S phase and acts as a platform for a large number of proteins involved in chromosome duplication at the replication fork. As duplication is completed, PCNA dissociates from chromatin, and thus, chromatin-bound PCNA levels are regulated during the cell cycle. Although the mechanism of PCNA loading has been extensively investigated, the unloading mechanism has remained unclear. Here, we show that Elg1, an alternative replication factor C protein, is required for the regulation of chromatin-bound PCNA levels. When Elg1 was depleted by small interfering RNA, chromatin-bound PCNA levels were extremely increased during the S phase. The number of PCNA foci, regions in the nucleus normally representing DNA replication sites, was increased and PCNA remained on chromatin after DNA replication. Various chromatin-associated protein levels on chromatin were affected, and chromatin loop size was increased. During mitosis, cells with aberrant chromosomes and lagging chromosomes were frequently detected. Our findings suggest that Elg1 has an important role in maintaining chromosome integrity by regulating PCNA levels on chromatin, thereby acting as a PCNA unloading factor.

Inhibition of DNA damage-induced apoptosis through Cdc7-mediated stabilization of Tob.

BACKGROUND: Preventing unnecessary cell death is essential for DNA-damaged cells to carry out the DNA repair process. RESULTS: Cdc7 inhibits the Cul4-DDB1(Cdt2)-dependent Tob degradation. CONCLUSION: Cdc7 enables mild DNA-damaged cells to keep their viability by competing with the Tob degradation system. SIGNIFICANCE: Cells deal with moderate DNA damage not only by cessation of the cell cycle but also through direct mediated pro-survival signaling. Cells respond to DNA damage by activating alternate signaling pathways that induce proliferation arrest or apoptosis. The correct balance between these two pathways is important for maintaining genomic integrity and preventing unnecessary cell death. The mechanism by which DNA-damaged cells escape from apoptosis during DNA repair is poorly understood. We show that the DNA replication-initiating kinase Cdc7 actively prevents unnecessary death in DNA-damaged cells. In response to mild DNA damage, Tob levels increase through both a transcriptional mechanism and protein stabilization, resulting in inhibition of pro-apoptotic signaling. Cells lacking Cdc7 expression undergo apoptosis after mild DNA damage, where Cul4-DDB1(Cdt2) induces Tob ubiquitination and subsequent degradation. Cdc7 phosphorylates and interacts with Tob to inhibit the Cul4-DDB1(Cdt2)-dependent Tob degradation. Thus, Cdc7 defines an essential pro-survival signaling pathway by contributing to stabilization of Tob, thereby the viability of DNA-damaged cells being maintained.

Dynamic recruitment of licensing factor Cdt1 to sites of DNA damage.

For genomic integrity to be maintained, the cell cycle and DNA damage responses must be linked. Cdt1, a G1-specific cell-cycle factor, is targeted for proteolysis by the Cul4-Ddb1(Cdt2) ubiquitin ligase following DNA damage. Using a laser nanosurgery microscope to generate spatially restricted DNA damage within the living cell nucleus, we show that Cdt1 is recruited onto damaged sites in G1 phase cells, within seconds of DNA damage induction. PCNA, Cdt2, Cul4, DDB1 and p21(Cip1) also accumulate rapidly to damaged sites. Cdt1 recruitment is PCNA-dependent, whereas PCNA and Cdt2 recruitment are independent of Cdt1. Fitting of fluorescence recovery after photobleaching profiles to an analytic reaction-diffusion model shows that Cdt1 and p21(Cip1) exhibit highly dynamic binding at the site of damage, whereas PCNA appears immobile. Cdt2 exhibits both a rapidly exchanging and an apparently immobile subpopulation. Our data suggest that PCNA provides an immobile binding interface for dynamic Cdt1 interactions at the site of damage, which leads to rapid Cdt1 recruitment to damaged DNA, preceding Cdt1 degradation.

Positively charged residues located downstream of PIP box, together with TD amino acids within PIP box, are important for CRL4(Cdt2) -mediated proteolysis.

PCNA links Cdt1 and p21 for proteolysis by Cul4-DDB1-Cdt2 (CRL4(Cdt2) ) in the S phase and after DNA damage in mammalian cells. However, other PCNA-interacting proteins, such as ligase I, are not targets of CRL4(Cdt2) . In this study, we created chimera constructs composed of Cdt1 and ligase I and examined how the proteolysis of PCNA-interacting proteins is regulated. Consistent with a recent report using the Xenopus egg system (Havens & Walter 2009), two amino acid elements are also required for degradation in HeLa cells: TD amino acid residues in the PIP box and the basic amino acid at +4 downstream of the PIP box. In addition, we demonstrate that a basic amino acid at +3 is also required for degradation and that an acidic amino acid residue following the basic amino acids abolishes the degradation. Electrostatic surface images suggest that the basic amino acid at +4 is involved in a contact with PCNA, while +3 position extending to opposite direction is important to create a positively charged surface. When all these required elements were introduced in ligase I peptide, the substituted form became degraded. Our results demonstrate that PCNA-dependent degron is strictly composed to avoid illegitimate destruction of PCNA-interacting proteins.

CRL4Cdt2 Ubiquitin Ligase, A Genome Caretaker Controlled by Cdt2 Binding to PCNA and DNA.

The ubiquitin ligase CRL4Cdt2 plays a vital role in preserving genomic integrity by regulating essential proteins during S phase and after DNA damage. Deregulation of CRL4Cdt2 during the cell cycle can cause DNA re-replication, which correlates with malignant transformation and tumor growth. CRL4Cdt2 regulates a broad spectrum of cell cycle substrates for ubiquitination and proteolysis, including Cdc10-dependent transcript 1 or Chromatin licensing and DNA replication factor 1 (Cdt1), histone H4K20 mono-methyltransferase (Set8) and cyclin-dependent kinase inhibitor 1 (p21), which regulate DNA replication. However, the mechanism it operates via its substrate receptor, Cdc10-dependent transcript 2 (Cdt2), is not fully understood. This review describes the essential features of the N-terminal and C-terminal parts of Cdt2 that regulate CRL4 ubiquitination activity, including the substrate recognition domain, intrinsically disordered region (IDR), phosphorylation sites, the PCNA-interacting protein-box (PIP) box motif and the DNA binding domain. Drugs targeting these specific domains of Cdt2 could have potential for the treatment of cancer.

Proliferating cell nuclear antigen-dependent rapid recruitment of Cdt1 and CRL4Cdt2 at DNA-damaged sites after UV irradiation in HeLa cells.

The licensing factor Cdt1 is degraded by CRL4(Cdt2) ubiquitin ligase dependent on proliferating cell nuclear antigen (PCNA) during S phase and when DNA damage is induced in G(1) phase. Association of both Cdt2 and PCNA with chromatin was observed in S phase and after UV irradiation. Here we used a micropore UV irradiation assay to examine Cdt2 accumulation at cyclobutane pyrimidine dimer-containing DNA-damaged sites in the process of Cdt1 degradation in HeLa cells. Cdt2, present in the nucleus throughout the cell cycle, accumulated rapidly at damaged DNA sites during G(1) phase. The recruitment of Cdt2 is dependent on prior PCNA chromatin binding because Cdt2 association was prevented when PCNA was silenced. Cdt1 was also recruited to damaged sites soon after UV irradiation through its PIP-box. As Cdt1 was degraded, the Cdt2 signal at damaged sites was reduced, but PCNA, cyclobutane pyrimidine dimer, and XPA (xeroderma pigmentosum, complementation group A) signals remained at the same levels. These findings suggest that Cdt1 degradation following UV irradiation occurs rapidly at damaged sites due to PCNA chromatin loading and the recruitment of Cdt1 and CRL4(Cdt2), before DNA damage repair is completed.

Polycomb-group complex 1 acts as an E3 ubiquitin ligase for Geminin to sustain hematopoietic stem cell activity.

Polycomb-group (PcG) genes encode multimeric nuclear protein complexes, PcG complex 1 and 2. PcG complex 2 was proved to induce transcription repression and to further methylate histone H3 at lysine-27 (H3K27). Subsequently PcG complex 1 is recruited through recognition of methylated H3K27 and maintains the transcription silencing by mediating monoubiquitination of histone H2A at lysine-119. Genetic evidence demonstrated a crucial role for PcG complex 1 in stem cells, and Bmi1, a member of PcG complex 1, was shown to sustain adult stem cells through direct repression of the INK4a locus encoding cyclin-dependent kinase inhibitor, p16CKI, and p19ARF. The molecular functions of PcG complex 1, however, remain insufficiently understood. In our study, deficiency of Rae28, a member of PcG complex 1, was found to impair ubiquitin-proteasome-mediated degradation of Geminin, an inhibitor of DNA replication licensing factor Cdt1, and to increase protein stability. The resultant accumulation of Geminin, based on evidence from retroviral transduction experiments, presumably eliminated hematopoietic stem cell activity in Rae28-deficient mice. Rae28 mediates recruiting Scmh1, which provides PcG complex 1 an interaction domain for Geminin. Moreover, PcG complex 1 acts as the E3 ubiquitin ligase for Geminin, as we demonstrated in vivo as well as in vitro by using purified recombinant PcG complex 1 reconstituted in insect cells. Our findings suggest that PcG complex 1 supports the activity of hematopoietic stem cells, in which high-level Geminin expression induces quiescence securing genome stability, by enhancing cycling capability and hematopoietic activity through direct regulation of Geminin.

CRL4Cdt2: Coupling Genome Stability to Ubiquitination.

The cullin-RING E3 ubiquitin ligase CRL4Cdt2 has emerged as a master regulator of genome stability, which targets key cell cycle proteins for proteolysis during S phase and after DNA damage. Recent advances shed light on how it couples ubiquitination to DNA synthesis, offering a new paradigm for substrate recognition: Cdt2 binds directly onto proliferating cell nuclear antigen (PCNA) loaded on DNA, which serves as a landing pad for the independent recruitment of the ubiquitin ligase and its substrates. Cyclin-dependent kinases (CDKs) and the ataxia telangiectasia and Rad3-related (ATR) kinase ensure accurate spatiotemporal regulation of CRL4Cdt2 under normal conditions and upon DNA damage. Deregulation of Cdt2 is evident in malignancies and was recently highlighted as a major target of oncogenic viruses, supporting the therapeutic targeting of the ligase as a promising anticancer strategy.

START-GAP2/DLC2 is localized in focal adhesions via its N-terminal region.

START-GAP2, also termed as DLC2, is a START domain-containing RhoGAP and a negative regulator of RhoA and Cdc42. Although it was reported as a tumor suppresser gene product, the molecular basis for function of START-GAP2 remains to be clarified. Here, we demonstrate that START-GAP2 is localized in focal adhesions through a "FAT (focal adhesion targeting)" region in the N-terminal half. START-GAP2 competes with START-GAP1/DLC1, another START domain-containing RhoGAP, in focal adhesion targeting. Moreover, the C-terminus of tensin2, one of focal adhesion components and reported to bind START-GAP1, also directly interacts with START-GAP2. These results suggest that START-GAP2 and START-GAP1 share the same molecular mechanism in targeting to focal adhesions.

Schizosaccharomyces pombe Snf2SR, a novel SNF2 family protein, interacts with Ran GTPase and modulates both RanGEF and RanGAP activities.

Snf2SR, a suppressor of rna1(ts), which is a temperature-sensitive mutation in Schizosaccharomyces pombe RanGAP (GTPase activating protein), possesses both the SNF2 and the helicase domains conserved in the chromatin remodeling SNF2 ATPase/helicase protein family. We have now clarified a function of Snf2SR. Snf2SR indeed showed DNA-stimulated ATPase activity, proving that it is a member of the SNF2 ATPase/helicase family. Consistent with this role, Snf2SR was localized in the nucleus and cell fractionation analysis revealed that Snf2SR was tightly associated with the nuclear matrix. The disruption of snf2SR(+) was detrimental for a cell proliferation of S. pombe. Snf2SR that did not enhance RanGAP activity by itself, but abolished histone-H3-mediated RanGAP inhibition, as previously reported for the histone H3 methyltransferase, Clr4, another rna1(ts) suppressor. In contrast to Clr4, Snf2SR directly bound to the GDP-bound form of the S. pombe Ran homologue Spi1 and enhanced the nucleotide exchange activity of Pim1, the S. pombe RanGEF (guanine nucleotide exchange factor). Over-expression of Spi1-G18V, a Ran GTPase mutant fixed in the GTP-bound form, was lethal to S. pombe Deltasnf2SR. Together, our results indicate that Snf2SR is involved in the Ran GTPase cycle in vivo.

Nuclear RanGAP is required for the heterochromatin assembly and is reciprocally regulated by histone H3 and Clr4 histone methyltransferase in Schizosaccharomyces pombe.

Although the Ran GTPase-activating protein RanGAP mainly functions in the cytoplasm, several lines of evidence indicate a nuclear function of RanGAP. We found that Schizosaccharomyces pombe RanGAP, SpRna1, bound the core of histone H3 (H3) and enhanced Clr4-mediated H3-lysine 9 (K9) methylation. This enhancement was not observed for methylation of the H3-tail containing K9 and was independent of SpRna1-RanGAP activity, suggesting that SpRna1 itself enhances Clr4-mediated H3-K9 methylation via H3. Although most SpRna1 is in the cytoplasm, some cofractionated with H3. Sprna1(ts) mutations caused decreases in Swi6 localization and H3-K9 methylation at all three heterochromatic regions of S. pombe. Thus, nuclear SpRna1 seems to be involved in heterochromatin assembly. All core histones bound SpRna1 and inhibited SpRna1-RanGAP activity. In contrast, Clr4 abolished the inhibitory effect of H3 on the RanGAP activity of SpRna1 but partially affected the other histones. SpRna1 formed a trimeric complex with H3 and Clr4, suggesting that nuclear SpRna1 is reciprocally regulated by histones, especially H3, and Clr4 on the chromatin to function for higher order chromatin assembly. We also found that SpRna1 formed a stable complex with Xpo1/Crm1 plus Ran-GTP, in the presence of H3.

A DNA-binding domain in the C-terminal region of Cdt2 enhances the DNA synthesis-coupled CRL4Cdt2 ubiquitin ligase activity for Cdt1.

The Cullin-RING ubiquitin ligase CRL4Cdt2 maintains genome integrity by mediating the cell cycle- and DNA damage-dependent degradation of proteins such as Cdt1, p21 and Set8. Human Cdt2 has two regions, a conserved N-terminal seven WD40 repeat region and a less conserved C-terminal region. Here, we showed that the N-terminal region is sufficient for complex formation with CRL4, but the C-terminal region is required for the full ubiquitin ligase activity. UV irradiation-induced polyubiquitination and degradation of Cdt1 were impaired in Cdt2 (N-terminus only)-expressing cells. Deletion and mutation analysis identified a domain in the C-terminal region that increased ubiquitination activity and displayed DNA-binding activity. The identified domain mediated binding to double-stranded DNA and showed higher affinity binding to single-stranded DNA. As the ligase activity of CRL4Cdt2 depends on proliferating cell nuclear antigen (PCNA) loading onto DNA, the present results suggest that the DNA-binding domain facilitates the CRL4Cdt2-mediated recognition and ubiquitination of substrates bound to PCNA on chromatin.

Author Correction: Phosphorylated HBO1 at UV irradiated sites is essential for nucleotide excision repair.

This corrects the article DOI: 10.1038/ncomms16102.

Direct binding of Cdt2 to PCNA is important for targeting the CRL4Cdt2 E3 ligase activity to Cdt1.

The CRL4Cdt2 ubiquitin ligase complex is an essential regulator of cell-cycle progression and genome stability, ubiquitinating substrates such as p21, Set8, and Cdt1, via a display of substrate degrons on proliferating cell nuclear antigens (PCNAs). Here, we examine the hierarchy of the ligase and substrate recruitment kinetics onto PCNA at sites of DNA replication. We demonstrate that the C-terminal end of Cdt2 bears a PCNA interaction protein motif (PIP box, Cdt2PIP), which is necessary and sufficient for the binding of Cdt2 to PCNA. Cdt2PIP binds PCNA directly with high affinity, two orders of magnitude tighter than the PIP box of Cdt1. X-ray crystallographic structures of PCNA bound to Cdt2PIP and Cdt1PIP show that the peptides occupy all three binding sites of the trimeric PCNA ring. Mutating Cdt2PIP weakens the interaction with PCNA, rendering CRL4Cdt2 less effective in Cdt1 ubiquitination and leading to defects in Cdt1 degradation. The molecular mechanism we present suggests a new paradigm for bringing substrates to the CRL4-type ligase, where the substrate receptor and substrates bind to a common multivalent docking platform to enable subsequent ubiquitination.

Architecture of the complete oxygen-sensing FixL-FixJ two-component signal transduction system.

The symbiotic nitrogen-fixing bacterium Bradyrhizobium japonicum is critical to the agro-industrial production of soybean because it enables the production of high yields of soybeans with little use of nitrogenous fertilizers. The FixL and FixJ two-component system (TCS) of this bacterium ensures that nitrogen fixation is only stimulated under conditions of low oxygen. When it is not bound to oxygen, the histidine kinase FixL undergoes autophosphorylation and transfers phosphate from adenosine triphosphate (ATP) to the response regulator FixJ, which, in turn, stimulates the expression of genes required for nitrogen fixation. We purified full-length B. japonicum FixL and FixJ proteins and defined their structures individually and in complex using small-angle x-ray scattering, crystallographic, and in silico modeling techniques. Comparison of active and inactive forms of FixL suggests that intramolecular signal transduction is driven by local changes in the sensor domain and in the coiled-coil region connecting the sensor and histidine kinase domains. We also found that FixJ exhibits conformational plasticity not only in the monomeric state but also in tetrameric complexes with FixL during phosphotransfer. This structural characterization of a complete TCS contributes both a mechanistic and evolutionary understanding to TCS signal relay, specifically in the context of the control of nitrogen fixation in root nodules.

Identification of novel suppressors for Mog1 implies its involvement in RNA metabolism, lipid metabolism and signal transduction.

Mog1 is conserved from yeast to mammal, but its function is obscure. We isolated yeast genes that rescued a temperature-sensitive death of S. cerevisiae Scmog1Delta, and of S. pombe Spmog1(ts). Scmog1Delta was rescued by Opi3p, a phospholipid N-methyltransferase, in addition to S. cerevisiae Ran-homologue Gsp1p, and a RanGDP binding protein Ntf2p. On the other hand, Spmog1(ts) was rescued by Cid13 that is a poly (A) polymerase specific for suc22(+) mRNA encoding a subunit of ribonucleotide reductase, Ssp1 that is a protein kinase involved in stress response pathway, and Crp79 that is required for mRNA export, in addition to Spi1, S. pombe Ran-homologue, and Nxt2, S. pombe homologue of Ntf2p. Consistent with the identification of those suppressors, lack of ScMog1p dislocates Opi3p from the nuclear membrane and all of Spmog1(ts) showed the nuclear accumulation of mRNA. Furthermore, SpMog1 was co-precipitated with Nxt2 and Cid13.

Two ubiquitin-conjugating enzymes, UbcP1/Ubc4 and UbcP4/Ubc11, have distinct functions for ubiquitination of mitotic cyclin.

Cell cycle events are regulated by sequential activation and inactivation of Cdk kinases. Mitotic exit is accomplished by the inactivation of mitotic Cdk kinase, which is mainly achieved by degradation of cyclins. The ubiquitin-proteasome system is involved in this process, requiring APC/C (anaphase-promoting complex/cyclosome) as a ubiquitin ligase. In Xenopus and clam oocytes, the ubiquitin-conjugating enzymes that function with APC/C have been identified as two proteins, UBC4 and UBCx/E2-C. Previously we reported that the fission yeast ubiquitin-conjugating enzyme UbcP4/Ubc11, a homologue of UBCx/E2-C, is required for mitotic transition. Here we show that the other fission yeast ubiquitin-conjugating enzyme, UbcP1/Ubc4, which is homologous to UBC4, is also required for mitotic transition in the same manner as UbcP4/Ubc11. Both ubiquitin-conjugating enzymes are essential for cell division and directly required for the degradation of mitotic cyclin Cdc13. They function nonredundantly in the ubiquitination of CDC13 because a defect in ubcP1/ubc4+ cannot be suppressed by high expression of UbcP4/Ubc11 and a defect in ubcP4/ubc11+ cannot be suppressed by high expression of UbcP1/Ubc4. In vivo analysis of the ubiquitinated state of Cdc13 shows that the ubiquitin chains on Cdc13 were short in ubcP1/ubc4 mutant cells while ubiquitinated Cdc13 was totally reduced in ubcP4/ubc11 mutant cells. Taken together, these results indicate that the two ubiquitin-conjugating enzymes play distinct and essential roles in the degradation of mitotic cyclin Cdc13, with the UbcP4/Ubc11-pathway initiating ubiquitination of Cdc13 and the UbcP1/Ubc4-pathway elongating the short ubiquitin chains on Cdc13.