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.

Background:It is unclear how DNA-damaging agents target cancer cells over normal somatic cells. Results: Arf/p53-dependent down-regulation of H2AX enables normal cells to survive after DNA damage. Conclusion: Transformed cells, which harbor mutations in either Arf or p53, are more sensitive to DNA-damaging agents. Significance: Cellular transformation renders cells more susceptible to some DNA-damaging agents.

The (6-4) photoproduct formed by ultraviolet light is known as an alkali-labile DNA lesion. Strand breaks occur at (6-4) photoproducts when UV-irradiated DNA is treated with hot alkali. We have analyzed the degradation reaction of this photoproduct under alkaline conditions using synthetic oligonucleotides. A tetramer, d(GT(6-4)TC), was prepared, and its degradation in 50 mM KOH at 60°C was monitored by high performance liquid chromatography. A single peak with a UV absorption spectrum similar to that of the starting material was detected after the reaction, and this compound was UV light in solar radiation is absorbed by nucleobases in DNA and induces photochemical reactions. The major forms of DNA damage by UV radiation are the cis-syn cyclobutane pyrimidine dimer (CPD) 1 and the pyrimidine(6-4)pyrimidone photoproduct ((6-4) photoproduct) formed between adjacent pyrimidine bases (1). The formation of these lesions not only induces mutations of genetic information (2) but also alters the chemical stability of DNA. In the case of the CPD formed at the sequences containing cytosine, the hydrolysis rate of the amino function is much higher than that of the normal cytosine base (3), which results in the C3 T transition (4 -6). The (6-4) photoproduct was originally identified as a UV-induced alkalisensitive lesion (7,8). Strand breaks occur at the sites of this photoproduct when UV-irradiated DNA is treated with hot alkali, and this procedure has been used to map the (6-4) photoproduct in defined DNA sequences at nucleotide resolution (9 -12). This alkali lability has also been applied for footprinting experiments to analyze protein-DNA interactions in vivo (13,14). While the reactions of other alkali-labile DNA damages such as the apurinic/apyrimidinic (AP) site (15) and the bleomycin-induced lesion (16) have been analyzed in detail, the mechanism of the strand break caused at the (6-4) photoproduct by the alkali treatment has not been elucidated so far. Franklin et al. (8) suggested the hydrolysis of the glycosyl bond of the 3Ј component of this photoproduct, but there have been no subsequent reports.The (6-4) photoproduct is reportedly more mutagenic than the CPD (17-19) and blocks replication without being bypassed by error-free translesion synthesis, whereas DNA polymerase correctly incorporates dATP opposite the CPD formed at the TT sequence (20, 21). To avoid mutagenesis and carcinogenesis by the (6-4) photoproduct, this damage must be removed by the nucleotide excision repair (NER) pathway in cells (22)(23)(24). No base excision repair (BER) enzyme is known for the (6-4) photoproduct, but if the photoproduct can be degraded artificially, this DNA may be repaired by the BER pathway, even in xeroderma pigmentosum patients lacking NER function. For such an application, it is important to characterize the chemical properties of the (6-4) photoproduct.In this paper we describe the analysis of the alkali degradation of DNA containing the (6-4) photoproduct using chemically synthesized oligonucleotides. A relati...

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DNA polymerase zeta (Pol ζ) participates in translesion synthesis (TLS) of DNA adducts that stall replication fork progression. Previous studies have led to the suggestion that the primary role of Pol ζ in TLS is to extend primers created when another DNA polymerase inserts nucleotides opposite lesions. Here we test the non-exclusive possibility that Pol ζ can sometimes perform TLS in the absence of any other polymerase. To do so, we quantified the efficiency with which S. cerevisiae Pol ζ bypasses abasic sites, cis-syn cyclobutane pyrimidine dimers and (6-4) photoproducts. In reactions containing dNTP concentrations that mimic those induced by DNA damage, a Pol ζ derivative with phenylalanine substituted for leucine 979 at the polymerase active site bypasses all three lesions at efficiencies between 27–73%. Wild-type Pol ζ also bypasses these lesions, with efficiencies that are lower and depend on the sequence context in which the lesion resides. The results are consistent with the hypothesis that, in addition to extending aberrant termini created by other DNA polymerases, Pol ζ has the potential to be the sole DNA polymerase involved in TLS.

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We have found that distamycin A can bind to DNA duplexes containing the (6-4) photoproduct, one of the major UV lesions in DNA, despite the changes, caused by photoproduct formation, in both the chemical structure of the base moiety and the local tertiary structure of the helix. A 20-mer duplex containing the target site, AATT.AATT, was designed, and then one of the TT sequences was changed to the (6-4) photoproduct. Distamycin binding to the photoproduct-containing duplex was detected by CD spectroscopy, whereas specific binding did not occur when the TT site was changed to a cyclobutane pyrimidine dimer, another type of UV lesion. Distamycin binding was analyzed in detail using 14-mer duplexes. Curve fitting of the CD titration data and induced CD difference spectra revealed that the binding stoichiometry changed from 1:1 to 2:1 with photoproduct formation. Melting curves of the drug-DNA complexes also supported this stoichiometry.

The (6-4) photoproduct is one of the major damaged bases produced by ultraviolet light in DNA. This lesion is known to be alkali-labile, and strand breaks occur at its sites when UV-irradiated DNA is treated with hot alkali. We have analyzed the product obtained by the alkali treatment of a dinucleoside monophosphate containing the (6-4) photoproduct, by HPLC, NMR spectroscopy, and mass spectrometry. We previously found that the N3-C4 bond of the 5' component was hydrolyzed by a mild alkali treatment, and the present study revealed that the following reaction was the hydrolysis of the glycosidic bond at the 3' component. The sugar moiety of this component was lost, even when a 3'-flanking nucleotide was not present. Glycosidic bond hydrolysis was also observed for a dimer and a trimer containing 5-methyl-2-pyrimidinone, which was used as an analog of the 3' component of the (6-4) photoproduct, and its mechanism was elucidated. Finally, the alkali treatment of a tetramer, d(GT(6-4)TC), yielded 2'-deoxycytidine 5'-monophosphate, while 2'-deoxyguanosine 3'-monophosphate was not detected. This result demonstrated the hydrolysis of the glycosidic bond at the 3' component of the (6-4) photoproduct and the subsequent strand break by β-elimination. It was also shown that the glycosidic bond at the 3' component of the Dewar valence isomer was more alkali-labile than that of the (6-4) photoproduct.