Search

The origin recognition complex (ORC), a multisubunit protein identified in Saccharomyces cerevisiae, binds to chromosomal replicators and is required for the initiation of cellular DNA replication. Complementary DNAs (cDNAs) encoding proteins related to the two largest subunits of ORC were cloned from various eukaryotes. The cDNAs encoding proteins related to S. cerevisiae Orc1 p were cloned from the budding yeast Kluyveromyces lactis, the fission yeast Schizosaccharomyces pombe, and human cells. These proteins show similarity to regulators of the S and M phases of the cell cycle. Genetic analysis of [ocr1.sup.+] from S. pombe reveals that it is essential for cell viability. The cDNAS encoding proteins related to S. cerevisiae Orc2p were cloned from Arabidopsis thaliana, Caenorhabditis elegans, and human cells. The human ORC-related proteins interact in vivo to form a complex. These studies suggest that ORC subunits are conserved and that the role of ORC is a general feature of eukaryotic DNA replication., The replicon model for the initiation of DNA replication postulates that for DNA synthesis to occur, an initiator protein is required for recognition of a specific replicator sequence in the [...]

Experimental analysis of the chromosome-derived DNA fragments (Hot DNA) shows that the DNA replication sites of Hot DNA of Escherichia coli are homologous to the Ter-A, -B and -C sites, suggesting that prevention of DNA replication fork at the Ter sites allows the RecBCD enzyme to enter the duplex DNA. The activities of Hot DNA are termination-dependent and -independent and require the Chi sequence, and mutations in the Chi sequence abolish Hot DNA activity. The dependence of Hot DNA activity on Chi sequence and its similarity to Ter sites indicates that Chi and Ter sequences have orientation and position similarities.

O6‐Methylguanines (O6‐meG), which are produced in DNA by the action of alkylating agents, are mutagenic and cytotoxic, and induce apoptosis in a mismatch repair (MMR) protein‐dependent manner. To understand the molecular mechanism of O6‐meG‐induced apoptosis, we performed functional analyses of FANCD2 and FANCI‐associated nuclease 1 (FAN1), which was identified as an interacting partner of MLH1. Immunoprecipitation analyses showed that FAN1 interacted with both MLH1 and MSH2 after treatment with N‐methyl‐N‐nitrosourea (MNU), indicating the formation of a FAN1‐MMR complex. In comparison with control cells, FAN1‐knockdown cells were more resistant to MNU, and the appearances of a sub‐G1 population and caspase‐9 activation were suppressed. FAN1 formed nuclear foci in an MLH1‐dependent manner after MNU treatment, and some were colocalized with both MLH1 foci and single‐stranded DNA (ssDNA) created at damaged sites. Under the same condition, FANCD2 also formed nuclear foci, although it was dispensable for the formation of FAN1 foci and ssDNA. MNU‐induced formation of ssDNA was dramatically suppressed in FAN1‐knockdown cells. We therefore propose that FAN1 is loaded on chromatin through the interaction with MLH1 and produces ssDNA by its exonuclease activity, which contributes to the activation of the DNA damage response followed by the induction of apoptosis triggered by O6‐meG. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) generated during cellular respiration oxidize various cellular constituents, which cause carcinogenesis. Because most studies on the role of ROS in carcinogenesis have mainly been performed using tumor-derived cell lines, which harbor various types of mutation, it has been difficult to determine the molecular details that lead to cancer formation. To overcome this difficulty, we established human-induced pluripotent stem cell lines in which the intracellular ROS levels are controlled at various differentiation stages by manipulating the ROS-yielding mitochondria. By introducing a specific amino acid substitution (I69E) into the succinate dehydrogenase complex, subunit C protein, a component of mitochondrial respiratory chain complex II, the ROS level increased considerably. When ROS-overproducing cells at the early stage of endoderm differentiation were subcutaneously inoculated into the backs of nude mice, we observed tumor formation. These tumor-initiating cells were subjected to a comprehensive analysis by RNA sequencing. It was revealed that tumor-initiating cells showed 27 upregulated transcripts compared with control cells. The newly identified genes include those coding for PAX8 and FOSB (transcription factors) as well as FGF22, whose expressions are known to increase in developing embryos. These results suggest that these genes may play a pivotal role in cancer formation at the very early stages of cell differentiation. [ABSTRACT FROM AUTHOR]

Folliculin-interacting protein 1 and 2 (FNIP1 and FNIP2) play critical roles in preventing renal malignancy through their association with the tumor suppressor FLCN. Mutations in FLCN are associated with Birt-Hogg-Dubé (BHD) syndrome, a rare disorder with increased risk of renal cancer. Recent studies indicated that FNIP1/FNIP2 double knockout mice display enlarged polycystic kidneys and renal carcinoma, which phenocopies FLCN knockout mice, suggesting that these two proteins function together to suppress renal cancer. However, the molecular mechanism functionally linking FNIP1/FNIP2 and FLCN remains largely elusive. Here, we demonstrated that FNIP2 protein is unstable and subjected to proteasome-dependent degradation via β-TRCP and Casein Kinase 1 (CK1)-directed ubiquitination in a nutrition-dependent manner. Degradation of FNIP2 leads to lysosomal dissociation of FLCN and subsequent lysosomal association of mTOR, which in turn promotes the proliferation of renal cancer cells. These results indicate that SCFβ-TRCP negatively regulates the FLCN complex by promoting FNIP degradation and provide molecular insight into the pathogenesis of BHD-associated renal cancer.

O6-Methylguanine, one of alkylated DNA bases, is especially mutagenic. Cells containing this lesion are eliminated by induction of apoptosis, associated with the function of mismatch repair (MMR) proteins. A retrovirus-mediated gene-trap mutagenesis was used to isolate new genes related to the induction of apoptosis, triggered by the treatment with an alkylating agent, N-methyl-N-nitrosourea (MNU). This report describes the identification of a novel gene, MAPO2 (O6- methylguanine-induced apoptosis 2), which is originally annotated as C1orf201. The MAPO2 gene is conserved among a wide variety of multicellular organisms and encodes a protein containing characteristic PxPxxY repeats. To elucidate the function of the gene product in the apoptosis pathway, a human cell line derived from HeLa MR cells, in which the MAPO2 gene was stably knocked down by expressing specific miRNA, was constructed. The knockdown cells grew at the same rate as HeLa MR, thus indicating that MAPO2 played no role in the cellular growth. After exposure to MNU, HeLa MR cells and the knockdown cells underwent cell cycle arrest at G2/M phase, however, the production of the sub-G1 population in the knockdown cells was significantly suppressed in comparison to that in HeLa MR cells. Moreover, the activation of BAK and caspase-3, and depolarization of mitochondrial membrane, hallmarks for the induction of apoptosis, were also suppressed in the knockdown cells. These results suggest that the MAPO2 gene product might positively contribute to the induction of apoptosis triggered by O6-methylguanine. [ABSTRACT FROM AUTHOR]

Abstract: When DNA is damaged by alkylating agents, apoptosis is induced to exclude cells carrying DNA lesions in order to prevent mutations and cancer. MAPO1, identified as a component involved in the induction of apoptosis, interacts with AMP-activated protein kinase (AMPK) and folliculin (FLCN). We herein report that MAPO1 is stabilized during the course of apoptosis, triggered by alkylation-induced O6-methylguanine in DNA. An immunoblotting analysis revealed that the amount of MAPO1 increased gradually after treatment with N-methyl-N-nitrosourea (MNU), although the level of mRNA for MAPO1 was unchanged. When cells were exposed to a proteasome inhibitor, MG132, the MAPO1 level significantly increased. On the other hand, application of a protein synthesis inhibitor, cycloheximide, caused a decrease in the MAPO1 content, implying that proteasome-mediated degradation is involved. In FLCN-knockdown cells, the MAPO1 level decreased, and no increases occurred even after MNU treatment. In contrast, stabilization of MAPO1 occurred in AMPKα-knockdown cells even without MNU treatment. While MAPO1 retains its ability to stably bind to FLCN, it dissociates gradually from AMPK after exposure to MNU. It seems that the proapoptotic function of MAPO1 may be regulated by AMPK and FLCN through stabilization of MAPO1 itself. [Copyright &y& Elsevier]

O 6 -Methylguanines (O 6 -meG), which are produced in DNA by the action of alkylating agents, are mutagenic and cytotoxic, and induce apoptosis in a mismatch repair (MMR) protein-dependent manner. To understand the molecular mechanism of O 6 -meG-induced apoptosis, we performed functional analyses of FANCD2 and FANCI-associated nuclease 1 (FAN1), which was identified as an interacting partner of MLH1. Immunoprecipitation analyses showed that FAN1 interacted with both MLH1 and MSH2 after treatment with N-methyl-N-nitrosourea (MNU), indicating the formation of a FAN1-MMR complex. In comparison with control cells, FAN1-knockdown cells were more resistant to MNU, and the appearances of a sub-G 1 population and caspase-9 activation were suppressed. FAN1 formed nuclear foci in an MLH1-dependent manner after MNU treatment, and some were colocalized with both MLH1 foci and single-stranded DNA (ssDNA) created at damaged sites. Under the same condition, FANCD2 also formed nuclear foci, although it was dispensable for the formation of FAN1 foci and ssDNA. MNU-induced formation of ssDNA was dramatically suppressed in FAN1-knockdown cells. We therefore propose that FAN1 is loaded on chromatin through the interaction with MLH1 and produces ssDNA by its exonuclease activity, which contributes to the activation of the DNA damage response followed by the induction of apoptosis triggered by O 6 -meG., (© 2020 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)