Joon-Kyu Lee

Assembly of the Cdc45-Mcm2–7-GINS complex in human cells requires the Ctf4/And-1, RecQL4, and Mcm10 proteins

In eukaryotes, the activation of the prereplicative complex and assembly of an active DNA unwinding complex are critical but poorly understood steps required for the initiation of DNA replication. In this report, we have used bimolecular fluorescence complementation assays in HeLa cells to examine the interactions between Cdc45, Mcm2–7, and the GINS complex (collectively called the CMG complex), which seem to play a key role in the formation and progression of replication forks. Interactions between the CMG components were observed only after the G1/S transition of the cell cycle and were abolished by treatment of cells with either a CDK inhibitor or siRNA against the Cdc7 kinase. Stable association of CMG required all three components of the CMG complex as well as RecQL4, Ctf4/And-1, and Mcm10. Surprisingly, depletion of TopBP1, a homologue of Dpb11 that plays an essential role in the chromatin loading of Cdc45 and GINS in yeast cells, did not significantly affect CMG complex formation. These results suggest that the proteins involved in the assembly of initiation complexes in human cells may differ somewhat from those in yeast systems.

The Schizosaccharomyces pombe origin recognition complex interacts with multiple AT-rich regions of the replication origin DNA by means of the AT-hook domains of the spOrc4 protein

The interaction between an origin sequence and the origin recognition complex (ORC), which is highly conserved in eukaryotes, is critical for the initiation of DNA replication. In this report, we have examined the interaction between the Schizosaccharomyces pombe (sp) autonomously replicating sequence 1 (ars1) and the spORC. For this purpose, we have purified the spORC containing all six subunits, a six-subunit complex containing the N-terminal-deleted spOrc4 subunit (spORCΔN-Orc4), and the spOrc4 subunit by using the baculovirus expression system. Wild-type spORC showed sequence-specific binding to ars1, and the spOrc4 protein alone showed the same DNA-binding properties as wild-type spORC. In contrast, the spORCΔN-Orc4 and the ΔN-spOrc4p alone did not bind significantly to ars1. These findings indicate that the N-terminal domain of the spOrc4 protein that contains multiple AT-hook motifs is essential for the ars1-binding activity. DNA-binding competition assays with fragments of ars1 and DNase I footprinting studies with full-length ars1 revealed that the spORC interacted with several AT-rich sequence regions of ars1. These DNA-binding properties of spORC correlate with the previously determined sequence requirements of the S. pombe ars1. These studies indicate that because of its unique Orc4 subunit, S. pombe uses a mechanism to recognize its origins different from that used by Saccharomyces cerevisiae.

The Cdc23 (Mcm10) protein is required for the phosphorylation of minichromosome maintenance complex by the Dfp1-Hsk1 kinase

Previous studies in Saccharomyces cerevisiae have defined an essential role for the Dbf4-Cdc7 kinase complex in the initiation of DNA replication presumably by phosphorylation of target proteins, such as the minichromosome maintenance (Mcm) complex. We have examined the phosphorylation of the Mcm complex by the Dfp1-Hsk1 kinase, the Schizosaccharomyces pombe homologue of Dbf4-Cdc7. In vitro, the purified Dfp1-Hsk1 kinase efficiently phosphorylated Mcm2p. In contrast, Mcm2p, present in the six-subunit Mcm complex, was a poor substrate of this kinase and required Cdc23p (homologue of Mcm10p) for efficient phosphorylation. In the presence of Cdc23p, Dfp1-Hsk1 phosphorylated the Mcm2p and Mcm4p subunits of the Mcm complex. Cdc23p interacted with both the Mcm complex and Dfp1-Hsk1 by selectively binding to the Mcm4/6/7 subunits and Dfp1p, respectively. The N terminus of Cdc23p was found to interact directly with Dfp1-Hsk1 and was essential for phosphorylation of the Mcm complex. Truncated derivatives of Cdc23p that complemented the temperature-sensitive phenotype of cdc23 mutant cells also stimulated the phosphorylation of Mcm complex, implying that this activity might be a critical role of Cdc23p in vivo. These results suggest that Cdc23p participates in the activation of prereplicative complex by recruiting the Dfp1-Hsk1 kinase and stimulating the phosphorylation of the Mcm complex.

ATR-dependent activation of p38 MAP kinase is responsible for apoptotic cell death in cells depleted of Cdc7.

Cdc7 is a serine/threonine kinase that plays essential roles in the initiation of eukaryotic DNA replication and checkpoint response. In previous studies, depletion of Cdc7 by small interfering RNA was shown to induce an abortive S phase that led to the cell cycle arrest in normal human fibroblasts and apoptotic cell death in various cancer cells. Here we report that stress-activated p38 MAP kinase was activated and responsible for apoptotic cell death in Cdc7-depleted HeLa cells. The activation of p38 MAP kinase in the Cdc7-depleted cells was shown to depend on ATR, a major sensor kinase for checkpoint or DNA damage responses. Only the p38 MAP kinase, and not the other stress-activated kinases such as JNK or ERK, was activated, and both caspase 8 and caspase 9 were activated for the induction of apoptosis. Activation of apoptosis in Cdc7-depleted cells was completely abolished in cells treated with small interfering RNA or an inhibitor of the p38 MAP kinase, suggesting that p38 MAP kinase activation was responsible for apoptotic cell death. Taken together, we suggest that the ATR-dependent activation of the p38 MAP kinase is a major signaling pathway that induces apoptotic cell death after depletion of Cdc7 in cancer cells.

Human Tim-Tipin complex affects the biochemical properties of the replicative DNA helicase and DNA polymerases

Tim (Timeless) and Tipin (Tim-interacting protein) form a stable heterodimeric complex that influences checkpoint responses and replication fork progression. We report that the Tim-Tipin complex interacts with essential replication fork proteins and affects their biochemical properties. The Tim-Tipin complex, reconstituted and purified using the baculovirus expression system, interacts directly with Mcm complexes and inhibits the single-stranded DNA-dependent ATPase activities of the Mcm2-7 and Mcm4/6/7 complexes, the DNA unwinding activity of the Mcm4/6/7 complex, and the DNA unwinding and ATPase activity of Cdc45-Mcm2-7-GINS complex, the presumed replicative DNA helicase in eukaryotes. Although stable interactions between Tim-Tipin and DNA polymerases (pols) were not observed in immunoprecipitation experiments with purified proteins, Tim was shown to interact with DNA pols α, δ, and ɛ in cells. Furthermore, the Tim-Tipin complex significantly stimulated the pol activities of DNA pols α, δ, and ɛ in vitro. The effects of Tim-Tipin on the catalytic activities of the Mcm complexes and DNA pols are mediated by the Tim protein alone, and distinct regions of the Tim protein are responsible for the inhibition of Mcm complex activities and stimulation of DNA pols. These results suggest that the Tim-Tipin complex might play a role in coupling DNA unwinding and DNA synthesis by directly affecting the catalytic activities of replication fork proteins.

Per3, a circadian gene, is required for Chk2 activation in human cells

MINT‐8052850: Chk2 (uniprotkb:O96017) physically interacts (MI:0915) with Per3 (uniprotkb:P56645) by anti bait coimmunoprecipitation (MI:0006)MINT‐8052875: Per3 (uniprotkb:P56645) physically interacts (MI:0914) with Chk2 (uniprotkb:O96017) and ATM (uniprotkb:Q13315) by anti tag coimmunoprecipitation (MI:0007)

RecQL4 is required for the association of Mcm10 and Ctf4 with replication origins in human cells

Though RecQL4 was shown to be essential for the initiation of DNA replication in mammalian cells, its role in initiation is poorly understood. Here, we show that RecQL4 is required for the origin binding of Mcm10 and Ctf4, and their physical interactions and association with replication origins are controlled by the concerted action of both CDK and DDK activities. Although RecQL4-dependent binding of Mcm10 and Ctf4 to chromatin can occur in the absence of pre-replicative complex, their association with replication origins requires the presence of the pre-replicative complex and CDK and DDK activities. Their association with replication origins and physical interactions are also targets of the DNA damage checkpoint pathways which prevent initiation of DNA replication at replication origins. Taken together, the RecQL4-dependent association of Mcm10 and Ctf4 with replication origins appears to be the first important step controlled by S phase promoting kinases and checkpoint pathways for the initiation of DNA replication in human cells.

Mimosine arrests the cell cycle prior to the onset of DNA replication by preventing the binding of human Ctf4/And-1 to chromatin via Hif-1α activation in HeLa cells.

Though the G1 checkpoint in mammalian cells has been known for decades, the molecular targets that prevent S-phase entry remain unknown. Mimosine is a rare plant amino acid that arrests the cell cycle in the G1 phase before entry into S phase. Here, we show that mimosine interrupts the binding of Ctf4 to chromatin, which is essential for the initiation of DNA replication in HeLa cells, and this effect is mediated by the Hif-1α-dependent increase in the level of p27. Depletion of Hif-1α results in an increased binding of Ctf4 to chromatin and the entry of cells into S phase even in the presence of mimosine. These results suggest that the binding of Ctf4 to chromatin is the target of the Hif-1α-dependent checkpoint pathway for cell cycle arrest in G1 phase. Although we observed Hif-1α-dependent arrest in mimosine-treated cells, it is possible that Ctf4 may act as a common target for G1 arrest in various other checkpoint pathways.

Meeting report: stem cell biology and epigenetics

The Genetics Society of Korea (GSK) had a symposium in the 68th Annual Meeting of the Korean Association of Biological Sciences (KABS) that was held during August 12–13 at Sogang University, Seoul, Korea. The symposium had two sessions on the two frontier fields in genetics; stem cell biology and epigenetics. The first session was on the stem cell biology that covered from the basics of the stem cell research to progresses in therapeutic approaches. Three active scientists presented their recent findings. The first speaker was Professor Dong Wook Han at the Konkuk University, Korea, with a title of ‘‘Inducing different stem cell fates by defined factors’’. Because the audience consists of diverse fields in genetics, he began his talk with nice introduction on the definition and sources of the stem cells. Recent advances in stem cell research have revealed that cell-type specific transcription factors can rest the somatic memory of differentiated cells and induce direct reprogramming into specific cell identity. The most remarkable achievement is that a combination of neural-specific transcription factors can induce a neural stem cell (NSC) fate on the fibroblasts. The experimental results showed that the induced neural stem cells (iNSCs) were similar to the wild type NSCs in cell morphology, gene expression, epigenetic features, differentiation potential, and cellular function. Thus, he posited that cell type-specific defined factors can induce specific stem cell identities from somatic cells. He also introduced his recent findings on the direct conversion of differentiated somatic cells into specific cellular identities such as hepatocytes. The second speaker professor, Hyuk-Jin Cha, at the Sogang University, Korea, gave a talk with a title of ‘‘Inhibition of pluripotent stem cell derived teratoma formation by small molecules’’. One of the obstacles in therapeutic application of the stem cells is the removal of residual undifferentiated pluripotent stem cells (PSCs) after differentiation. Because the undifferentiated PSCs can proliferate unlimitedly to form a tumor mass, it is critical to sort completely out the residual undifferentiated PSCs for tumor-free cell therapy. Several approaches have been suggested to eliminate the remaining undifferentiated cells, including the introduction of suicide gene, immunedepletion and introducing cytotoxic antibodies. However, the rigorous sorting techniques are still far behind in isolating the differentiated cells purely and have yet reached at the level of clinically viable strategy to eliminate teratoma formation. PSCs are highly susceptible to apoptotic stimuli such as reactive oxygen species and DNA damage stress which can induce genetic alteration to the differentiated cells. Thus, high responsiveness to apoptosis can be considered to be one of the protective mechanisms to retain genetic integrity during differentiation. Dr. Cha’s group analyzed the proand anti-apoptotic genes in human PSCs (hPSCs) and found that 22 pro-apoptotic genes and 10 antiapoptotic genes were highly expressed in undifferentiated PSCs. Of interest, inhibition of two anti-apoptotic factors (BIRC5 and BCL10) by small molecule could significantly induce selective cell death of PSCs but not differentiated J.-K. Lee Department of Biology Education, Seoul National University, Seoul 152-742, Korea

Depletion of the pre‐RC proteins induces Chk1/Chk2 independent checkpoint responses and apoptotic cell death in HeLa cells

Abstract The initiation of eukaryotic DNA replication requires assembly of the pre‐replicative complex (Pre‐RC) through the concerted action of Orc, Cdc6, Cdt1 and Mcm2–7 complex during G1 phase. The pre‐RC assembly licenses individual replication origins for the initiation of DNA replication and sufficient number of the pre‐RC is essential for proper progression of S phase. However, it is not well known how cells recognize the completion of the pre‐RC assembly before G1‐S transition. In order to understand the cellular responses to the defects in pre‐RC assembly, we depleted the known components of pre‐RC proteins using the small interference RNAs in HeLa cells. Although the defects of pre‐RC assembly by the depletion of the pre‐RC proteins such as Orc2, Cdt1, Mcm2 & Mcm10 did not elicit the activation of Chk1‐ or Chk2‐dependent checkpoint pathways, these cells still showed significant decrease in the cellular level of Cdc25A proteins. These results suggests that a novel checkpoint pathway exist in HeLa cells, which is not dependent upon Chk1 or Chk2 proteins and play essential roles in the cellular responses to the defects in the pre‐RC assembly. Also, among those four proteins tested in this study, the depletion of Mcm 10 and Cdt1 proteins significantly increased the apoptotic cell death in HeLa cells, suggesting that these proteins not only play roles in the pre‐RC assembly, but also are involved in the checkpoint responses to the defects in the pre‐RC assembly.