S(N)1-type alkylating agents that produce cytotoxic O(6)-methyl-G (O(6)-meG) DNA adducts induce cell cycle arrest and apoptosis in a manner requiring the DNA mismatch repair (MMR) proteins MutSalpha and MutLalpha. Here, we show that checkpoint signaling in response to DNA methylation occurs during S phase and requires DNA replication that gives rise to O(6)-meG/T mispairs. DNA binding studies reveal that MutSalpha specifically recognizes O(6)-meG/T mispairs, but not O(6)-meG/C. In an in vitro assay, ATR-ATRIP, but not RPA, is preferentially recruited to O(6)-meG/T mismatches in a MutSalpha- and MutLalpha-dependent manner. Furthermore, ATR kinase is activated to phosphorylate Chk1 in the presence of O(6)-meG/T mispairs and MMR proteins. These results suggest that MMR proteins can act as direct sensors of methylation damage and help recruit ATR-ATRIP to sites of cytotoxic O(6)-meG adducts to initiate ATR checkpoint signaling.

During tumorigenesis, cells acquire immortality in association with the development of genomic instability. However, it is still elusive how genomic instability spontaneously generates during the process of tumorigenesis. Here, we show that precancerous DNA lesions induced by oncogene acceleration, which induce situations identical to the initial stages of cancer development, trigger tetraploidy/aneuploidy generation in association with mitotic aberration. Although oncogene acceleration primarily induces DNA replication stress and the resulting lesions in the S phase, these lesions are carried over into the M phase and cause cytokinesis failure and genomic instability. Unlike directly induced DNA double-strand breaks, DNA replication stress-associated lesions are cryptogenic and pass through cell-cycle checkpoints due to limited and ineffective activation of checkpoint factors. Furthermore, since damaged M-phase cells still progress in mitotic steps, these cells result in chromosomal mis-segregation, cytokinesis failure and the resulting tetraploidy generation. Thus, our results reveal a process of genomic instability generation triggered by precancerous DNA replication stress.

Normal cells, both in vivo and in vitro, become quiescent after serial cell proliferation. During this process, cells can develop immortality with genomic instability, although the mechanisms by which this is regulated are unclear. Here, we show that a growth-arrested cellular status is produced by the down-regulation of histone H2AX in normal cells. Normal mouse embryonic fibroblast cells preserve an H2AX diminished quiescent status through p53 regulation and stable-diploidy maintenance. However, such quiescence is abrogated under continuous growth stimulation, inducing DNA replication stress. Because DNA replication stress-associated lesions are cryptogenic and capable of mediating chromosome-bridge formation and cytokinesis failure, this results in tetraploidization. Arf/p53 module-mutation is induced during tetraploidization with the resulting H2AX recovery and immortality acquisition. Thus, although cellular homeostasis is preserved under quiescence with stable diploidy, tetraploidization induced under growth stimulation disrupts the homeostasis and triggers immortality acquisition.

We performed molecular dynamics (MD) simulation that includes multidisciplinary characteristics from synthesis to mechanical properties of epoxy resin. First, to reproduce the actual chemical reaction between matrix and curing agents, we conducted curing simulation wherein the activation energy and heat of formation are considered for the chemical reaction. Subsequently, we performed MD simulations using cross-linked structure obtained from curing simulation to derive density and Young's modulus. Results indicated that crosslinked structures involving both activation energy and heat of formation could reproduce experiment results that are evaluated using differential scanning calorimetry (DSC) measurements and mechanical tests. The simulated results imply that electrostatic interaction plays an important role in Young's modulus. The density of the hydrogen bond between the oxygen of the hydroxyl group and the hydrogen atom is a key factor for the difference in Young's modulus for each base resin. These findings confirm that MD simulation is a potential alternative to experiments for the appropriate material selection of epoxy resin.

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The loss of DNA mismatch repair (MMR) is responsible for hereditary nonpolyposis colorectal cancer and a subset of sporadic tumors. Acquired resistance or tolerance to some anti-cancer drugs occurs when MMR function is impaired. 5-Fluorouracil (FU), an anti-cancer drug used in the treatment of advanced colorectal and other cancers, and its metabolites are incorporated into RNA and DNA and inhibit thymidylate synthase resulting in depletion of dTTP and incorporation in DNA of uracil. Although the MMR deficiency has been implicated in tolerance to FU, the mechanism of cell killing remains unclear. Here, we examine the cellular response to fluorodeoxyuridine (FdU) and the role of the MMR system. After brief exposure of cells to low doses of FdU, MMR mediates DNA damage signaling during S-phase and triggers arrest in G2/M in the first cell cycle in a manner requiring MutSα, MutLα, and DNA replication. Cell cycle arrest is mediated by ATR kinase and results in phosphorylation of Chk1 and SMC1. MutSα binds FdU:G mispairs in vitro consistent with its being a DNA damage sensor. Prolonged treatment with FdU results in an irreversible arrest in G2 that is independent of MMR status and leads to the accumulation of DNA lesions that are targeted by the base excision repair (BER) pathway. Thus, MMR can act as a direct sensor of FdU-mediated DNA lesions eliciting cell cycle arrest via the ATR/Chk1 pathway. However, at higher levels of damage, other damage surveillance pathways such as BER also play important roles. KeywordsMISMATCH REPAIR; 5-FLUOURACIL; DNA DAMAGE; ATR; COLORECTAL CANCER DNA mismatch repair (MMR) is a highly conserved repair pathway that plays an important role in the detection and correction of DNA mismatches created during replication and recombination. Inactivating mutations in MMR genes cause a greatly increased rate of spontaneous mutation and are the underlying defect in hereditary nonpolyposis colorectal cancer (HNPCC). In addition, MMR defects are associated with a significant proportion of sporadic cancers. Two key MMR proteins, MutS and MutL in prokaryotes and their eukaryotic counterparts, mediate a number of functions in maintaining genome integrity including the correction of DNA biosynthetic errors, suppression of illegitimate recombination, and participation in the cellular response to certain types of DNA damage [Schofield and Hsieh, 2003;Kunkel and Erie, 2005;Iyer et al., 2006]. In its role in correcting mispaired bases arising during replication, MutS targets DNA mismatches and together with MutL, licenses the In mammalian cells, the MMR system is also implicated in the cellular response to DNA damage resulting from exposure to S N 1 DNA methylating agents, 6-thioguanine, fluoropyrimidines (FPs), cisplatin, reactive oxygen species, ultraviolet light, and some environmental carcinogens [Iyer et al., 2006]. Cell killing mediated by S N 1 alkylators such as temozolomide that is commonly used in cancer chemotherapy requires functional MMR proteins. Tolerance or resistance to drug treatme...

ZPT2-2 is a DNA-binding protein of petunia that contains two canonical TFIIIA-type zinc finger motifs separated by a long linker. We previously reported that ZPT2-2 bound to two separate AGT core sites, with each zinc finger making contact with each core site. Here we present our further characterization of ZPT2-2 by using selected and amplified binding sequence imprinting and surface plasmon resonance analyses; together, these assays revealed some unusual features of the interaction between ZPT2-2 and DNA. These experiments allowed us to conclude that 1) the optimal binding sequence for the N-terminal zinc finger is AGC(T), and that of the C-terminal one is CAGT; 2) multiple arrangements of the two core sites accommodate binding; and 3) the spacing between the two core sites affects the binding affinity. In light of these observations, we propose a new model for the DNA-ZPT2-2 interaction. Further, consistent with this model, a high affinity binding site for ZPT2-2 was found in the promoter region of the ZPT2-2 gene. This site may serve as a cis-element for the autoregulation of ZPT2-2 gene expression.The TFIIIA (Cys 2 /His 2 )-type zinc finger proteins, first discovered in transcription factor IIIA of Xenopus (1), represent an important class of eukaryotic transcription factors. To date, numerous genes have been found that encode this type of zinc finger motif, and many of their products have been implicated in various regulatory roles. The TFIIIA-type zinc finger tetrahedrally coordinates a zinc atom to form a compact structure that interacts with the major groove of DNA in a sequencespecific manner. Generally, in animals, multiple zinc finger motifs are present as tandem arrays that are separated by a conserved short sequence known as an HC link (2). These cluster-type zinc finger proteins interact with contiguous sets of triplet sequences, with each zinc finger making contact with a triplet. In Sp1, Krox20, Zif268, and GAGA, specific amino acid residues in the ␣-helical region of the DNA binding surface have been shown to interact with specific nucleotides in target sequences (3-6).The EPF family is a subfamily of TFIIIA-type zinc finger proteins of plants. Members of the EPF family have been implicated in floral organ-specific (7-9) and stress-responsive (10) transcriptional regulation and other regulatory processes (11). The proteins of this family are characterized by the long (19 -65 amino acids) linkers of various lengths that separate the zinc fingers. Moreover, the zinc finger motif itself contains a highly conserved sequence, QALGGH (12, 13), which is located within the region that corresponds to the DNA-contacting surface of TFIIIA-type zinc finger proteins of animals (3-6).Our previous DNA binding studies showed that ZPT2-2, with a linker of 44 amino acids between the two fingers, interacted with two tandemly repeated AGT core sites, which were separated by 10 bp, 1 at a dissociation constant (K d ) of 120 nM (14). The binding affinity was sensitive to the spacing between the two core sites. Anoth...

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