Yukio Ishimi

Translocation and Stability of Replicative DNA Helicases upon Encountering DNA-Protein Cross-links

Background: DNA-protein cross-links (DPCs) are formed by DNA-damaging agents. Results: DPCs on the translocating strand but not on the nontranslocating strand block hexameric replicative helicases in a size-dependent manner. Stalled helicases dissociate from DNA with a half-life of 15–36 min. Conclusion: DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. Significance: Reversible and irreversible protein roadblocks may have distinct effects on replisomes. DNA-protein cross-links (DPCs) are formed when cells are exposed to various DNA-damaging agents. Because DPCs are extremely large, steric hindrance conferred by DPCs is likely to affect many aspects of DNA transactions. In DNA replication, DPCs are first encountered by the replicative helicase that moves at the head of the replisome. However, little is known about how replicative helicases respond to covalently immobilized protein roadblocks. In the present study we elucidated the effect of DPCs on the DNA unwinding reaction of hexameric replicative helicases in vitro using defined DPC substrates. DPCs on the translocating strand but not on the nontranslocating strand impeded the progression of the helicases including the phage T7 gene 4 protein, simian virus 40 large T antigen, Escherichia coli DnaB protein, and human minichromosome maintenance Mcm467 subcomplex. The impediment varied with the size of the cross-linked proteins, with a threshold size for clearance of 5.0–14.1 kDa. These results indicate that the central channel of the dynamically translocating hexameric ring helicases can accommodate only small proteins and that all of the helicases tested use the steric exclusion mechanism to unwind duplex DNA. These results further suggest that DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. The helicases stalled by DPC had limited stability and dissociated from DNA with a half-life of 15–36 min. The implications of the results are discussed in relation to the distinct stabilities of replisomes that encounter tight but reversible DNA-protein complexes and irreversible DPC roadblocks.

Proteasome inhibitors remarkably prevent translesion replication in cancer cells but not normal cells

When a replicative DNA polymerase encounters a lesion on the template strand and stalls, it is replaced with another polymerase(s) with low processivity that bypasses the lesion to continue DNA synthesis. This phenomenon is known as translesion replication or replicative bypass. Failing this, the cell is increasingly likely to undergo apoptosis. In this study, we found that proteasome inhibitors prevent translesion replication in human cancer cells but not in normal cells. Three proteasome inhibitors, MG‐132, lactacystin, and MG‐262, inhibited UV‐induced translesion replication in a wide range of cancer cell lines, including HeLa, HGC‐27, MCF‐7, HepG2, WiDr, a malignant melanoma, an acute lymphoblastic leukemia, and a multiple myeloma cell line; irrespective of cell origin, histological type, or p53 status. In contrast, these inhibitors had little or no influence on normal fibroblasts (NB1RGB and TIG‐1) or a normal liver mesenchymal (LI90) cell line. Among the DNA‐damaging antineoplastic agents, cisplatin caused a UV‐type translesion reaction; the proteasome inhibitors delayed cisplatin‐induced translesion replication in cancer cell lines but had only a weak effect on normal cell lines. Therefore, translesion replication would be an effective target of proteasome inhibitors for cancer chemotherapy by which cancer cells can be efficiently sensitized to DNA‐damaging antineoplastic agents, such as cisplatin. (Cancer Sci 2008; 99: 863–871)

Inhibition of DNA binding of MCM2-7 complex by phosphorylation with cyclin-dependent kinases.

Cyclin-dependent kinase (CDK) that plays a central role in preventing re-replication of DNA phosphorylates several replication proteins to inactivate them. MCM4 in MCM2-7 and RPA2 in RPA are phosphorylated with CDK in vivo. There are inversed correlations between the phosphorylation of these proteins and their chromatin binding. Here, we examined in vitro phosphorylation of human replication proteins of MCM2-7, RPA, TRESLIN, CDC45 and RECQL4 with CDK2/cyclinE, CDK2/cyclinA, CDK1/cyclinB, CHK1, CHK2 and CDC7/DBF4 kinases. MCM4, RPA2, TRESLIN and RECQL4 were phosphorylated with CDKs. Effect of the phosphorylation by CDK2/cyclinA on DNA-binding abilities of MCM2-7 and RPA was examined by gel-shift analysis. The phosphorylation of RPA did not affect its DNA-binding ability but that of MCM4 inhibited the ability of MCM2-7. Change of six amino acids of serine and threonine to alanines in the amino-terminal region of MCM4 rendered the mutant MCM2-7 insensitive to the inhibition with CDK. These biochemical data suggest that phosphorylation of MCM4 at these sites by CDK plays a direct role in dislodging MCM2-7 from chromatin and/or preventing re-loading of the complex to chromatin.

Interaction of heliquinomycin with single-stranded DNA inhibits MCM4/6/7 helicase.

The antibiotic heliquinomycin inhibited cellular DNA replication at IC(50) of 2.5 µM without affecting level of chromatin-bound MCM4 and without activating the DNA replication stress checkpoint system, suggesting that heliquinomycin perturbs DNA replication mainly by inhibiting the activity of replicative DNA helicase that unwinds DNA duplex at replication forks. Among the DNA helicases involved in DNA replication, DNA helicase B was inhibited by heliquinomycin at IC(50) of 4.3 µM and RECQL4 helicase at IC(50) of 14 µM; these values are higher than that of MCM4/6/7 helicase (2.5 µM). These results suggest that heliquinomycin mainly targets actions of the replicative DNA helicases. Gel-retardation experiment indicates that heliquinomycin binds to single-stranded DNA. The single-stranded DNA-binding ability of MCM4/6/7 was affected in the presence of heliquinomycin. The data suggest that heliquinomycin inhibits the DNA helicase activity of MCM4/6/7 complex by stabilizing its interaction with single-stranded DNA.

Protein interaction and cellular localization of human CDC45

CDC45, which plays a role in eukaryotic DNA replication, is a member of the CMG (CDC45/MCM2-7/GINS) complex that is thought to function as a replicative DNA helicase. However, the biochemical properties of CDC45 are not fully understood. We systematically examined the interactions of human CDC45 with MCM2-7, GINS and other replication proteins by immunoprecipitation. We found that CDC45 can directly interact with all MCM2-7 proteins; with PSF2, PSF3 and SLD5 in GINS subunits; and with replication protein A2 (RPA2), AND-1 and topoisomerase 2-binding protein 1. These results are consistent with the notion that CDC45 plays a role in progression of DNA replication forks. Experiments using antibodies against CDC45 show that the level of CDC45 recovered from the Triton-insoluble chromatin-containing fraction is peaked at middle of S phase in synchronized HeLa cells. However, incubation of the Triton-insoluble fraction with nucleases resulted in recovery of less than half the amount of CDC45 in the nuclease-sensitive fraction; this result is in contrast with RPA1 and proliferating cell nuclear antigen distribution. These results indicate that a considerable portion of CDC45 localizes in a region other than the DNA replication forks in nuclei or it localizes on the replication forks but it is not fractionated with the fork proteins owing to its tight association with presumably nuclear scaffolds.

Effect of an MCM4 mutation that causes tumours in mouse on human MCM4/6/7 complex formation.

It has been reported that a point mutation of minichromosome maintenance (MCM)4 causes mammary carcinoma, and it deregulates DNA replication to produce abnormal chromosome structures. To understand the effect of this mutation at level of MCM2-7 interaction, we examined the effect of the same mutation of human MCM4 on the complex formation with MCM6 and MCM7 in insect cells. Human MCM4/6/7 complexes containing the mutated MCM4 were formed, but the hexameric complex formation was not evident in comparison with those containing wild-type MCM4. In binary expression of MCM4 and MCM6, decreased levels of MCM6 were recovered with the mutated MCM4, compared with wild-type MCM4. These results suggest that this mutation of MCM4 perturbs proper interaction with MCM6 to affect complex formation of MCM4/6/7 that is a core structure of MCM2-7 complex. Consistent with this notion, nuclear localization and MCM complex formation of forcedly expressed MCM4 in human cells are affected by this mutation. Thus, the defect of this mutant MCM4 in interacting with MCM6 may generate a decreased level of chromatin binding of MCM2-7 complex.

Interaction of human minichromosome maintenance protein-binding protein with minichromosome maintenance 2–7

It has been reported that minichromosome maintenance protein‐binding protein (MCM‐BP) functions in the formation of the pre‐replication complex, unloading of minichromosome maintenance (MCM)2–7 from chromatin in late S phase, and formation of the cohesion complex by interacting with MCM3–7 proteins, suggesting that MCM‐BP functions in several different reactions during the cell cycle. Here, we examined the interaction of human MCM‐BP with MCM2–7 and structural maintenance of chromosome 3 in synchronized HeLa cells by immunoprecipitation. The results show that MCM‐BP mainly interacts with MCM7 in the Triton‐soluble fraction from S phase and G2 phase cells, and it also interacts with structural maintenance of chromosome 3 in the fraction from G2 phase cells. In vitro studies show that MCM‐BP disassembles MCM2–7 bound to DNA with a fork‐like structure by interacting with MCM3, MCM5, and MCM7. These results suggest that MCM‐BP functions in disassembling MCM2–7 on chromatin during S phase and G2 phase by interacting with MCM3, MCM5, and MCM7.

G364R mutation of MCM4 detected in human skin cancer cells affects DNA helicase activity of MCM4/6/7 complex.

A number of gene mutations are detected in cells derived from human cancer tissues, but roles of these mutations in cancer cell development are largely unknown. We examined G364R mutation of MCM4 detected in human skin cancer cells. Formation of MCM4/6/7 complex is not affected by the mutation. Consistent with this notion, the binding to MCM6 is comparable between the mutant MCM4 and wild-type MCM4. Nuclear localization of this mutant MCM4 expressed in HeLa cells supports this conclusion. Purified MCM4/6/7 complex containing the G364R MCM4 exhibited similar levels of single-stranded DNA binding and ATPase activities to the complex containing wild-type MCM4. However, the mutant complex showed only 30-50% of DNA helicase activity of the wild-type complex. When G364R MCM4 was expressed in HeLa cells, it was fractionated into nuclease-sensitive chromatin fraction, similar to wild-type MCM4. These results suggest that this mutation does not affect assembly of MCM2-7 complex on replication origins but it interferes some step at function of MCM2-7 helicase. Thus, this mutation may contribute to cancer cell development by disturbing DNA replication.

An MCM4 mutation detected in cancer cells affects MCM4/6/7 complex formation

An MCM4 mutation detected in human cancer cells from endometrium was characterized. The mutation of G486D is located within MCM-box and the glycine at 486 in human MCM4 is conserved in Saccharomyces cerevisiae MCM4 and Sulfolobus solfataricus MCM. This MCM4 mutation affected human MCM4/6/7 complex formation, since the complex containing the mutant MCM4 protein is unstable and the mutant MCM4 protein is tend to be degraded. It is likely that the MCM4 mutation affects the interaction with MCM7 to destabilize the MCM4/6/7 complex. Cells with abnormal nuclear morphology were detected when the mutant MCM4 was expressed in HeLa cells, suggesting that DNA replication was perturbed in the presence of the mutant MCM4. Role of the conserved amino acid in MCM4 function is discussed.

Binding of Human MCM-BP with MCM2-7 Proteins

MCM2-7 proteins play essential roles in DNA replication in eukaryotic cells, probably by acting as a replicative DNA helicase that unwinds DNA duplexes at replication forks (Bell & Dutta, 2002; Forsburg, 2004; Masai et al., 2010). Several lines of evidences suggest that MCM2-7 hexameric complexes assembled on the replication origins are converted to an active form with the assistance of the CDC7, CDC45 and GINS complex (Moyer et al., 2006; Gambus et al., 2006). MCM-BP, which has been identified from human cells as a protein that binds to MCM6 and MCM7 proteins, has amino acids sequences homologous to MCM2-7 (Sakwe et al. 2007). The results in this report indicate that MCM-BP replaces MCM2 in MCM2-7 complex and it binds to a replication origin in HeLa cells, suggesting that the MCM complex containing the MCM-BP may play a role in the initiation of DNA replication. It has also been indicated that downregulation of MCM-BP affects chromatin binding of MCM4. Recently it has been reported that Arabidopsis thaliana ETG1, which has been identified as an E2F target gene, is a homolog of MCM-BP (Takahashi et al., 2008). ETG1 protein is required for efficient DNA replication. Depletion of ETG1 results in inhibition of DNA replication and G2 arrest. Under these conditions, the G2 checkpoint system is induced. The report by Takahashi et al. (2010) indicates that ETG1 is involved in sister chromatid cohesion which is required for post-replicative homologous recombination repair. More recently, it has been reported that Xenopus MCM-BP regulates unloading of the MCM2-7 complex from chromatin in the late S phase by interacting with MCM7 (Nishiyama et al., 2011). These evidences suggest a possibility that MCM-BP may interact with the MCM2-7 complex at the replication forks to regulate the chromatin binding of the complex. Such interaction may be required for establishment of the cohesin complex at the forks. Here we examined biochemical properties of human MCM-BP. First, we found that human MCM-BP can bind all the human MCM2-7 proteins when the MCM-BP and one of the MCM2-7 proteins are co-expressed in insect cells. However, the interaction of MCM-BP with MCM7 was mainly detected when all the MCM2-7 and MCM-BP were co-expressed at the same time. In HeLa cells, MCM-BP was mainly recovered in a Triton-soluble fraction, suggesting that it does not stably bind to chromatin. A small portion of MCM-BP in this fraction was bound to MCM4, MCM5, MCM6 and MCM7 proteins. These results suggest that MCM-BP is not a constituent of pre-RC and it exhibits its functions by interacting with