p53 Specifically Binds Triplex DNA In Vitro and in Cells.

Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA recognition are core attributes of the p53 protein. The focus of this work is the structure-specific binding of p53 to DNA containing triplex-forming sequences in vitro and in cells and the effect on p53-driven transcription. This is the first DNA binding study of full-length p53 and its deletion variants to both intermolecular and intramolecular T.A.T triplexes. We demonstrate that the interaction of p53 with intermolecular T.A.T triplex is comparable to the recognition of CTG-hairpin non-B DNA structure. Using deletion mutants we determined the C-terminal DNA binding domain of p53 to be crucial for triplex recognition. Furthermore, strong p53 recognition of intramolecular T.A.T triplexes (H-DNA), stabilized by negative superhelicity in plasmid DNA, was detected by competition and immunoprecipitation experiments, and visualized by AFM. Moreover, chromatin immunoprecipitation revealed p53 binding T.A.T forming sequence in vivo. Enhanced reporter transactivation by p53 on insertion of triplex forming sequence into plasmid with p53 consensus sequence was observed by luciferase reporter assays. In-silico scan of human regulatory regions for the simultaneous presence of both consensus sequence and T.A.T motifs identified a set of candidate p53 target genes and p53-dependent activation of several of them (ABCG5, ENOX1, INSR, MCC, NFAT5) was confirmed by RT-qPCR. Our results show that T.A.T triplex comprises a new class of p53 binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in cells. The contribution of p53 DNA structure-dependent binding to the regulation of transcription is discussed.

Photochemical probing of the B--a conformational transition in a linearized pUC19 DNA and its polylinker region.

We induced the B-to-A conformational transition by ethanol in a linearized pUC19 DNA. A primer extension method was used in combination with UV light irradiation to follow the transition, based on pausing of DNA synthesis due to the presence of damaged bases in the template. Primer extension data highly correlated with the results of another method monitoring the B-A transition, i.e. inhibition of restriction endonuclease cleavage of UV light-irradiated DNA. Primer extension enabled us to locate damaged nucleotides within the region of interest. Most damaged nucleotides were located in B-form trimers, exclusively containing both pyrimidine bases (TTC, TCT, CTC, and CTT), and in a cytosine tetramer. The amount of damaged bases decreased in the course of B-A transition. Some of the damage even disappeared in the A-form, which mainly concerns the C(4) and C(3) blocks. The cleavage was nearly restored in the A-form within this region (Eco88I). On the contrary the decrease of damage was less significant with thymine dimers, only dropping to 50-60% of the B-form level. Consequently, the cleavage with EcoRI and HindIII remained mostly as before the transition (75% and 60% of uncleaved DNA preserved). We found significant differences in the B- and A-form pattern of UV light-damaged bases within the same region (polylinker) of DNA embedded within long (plasmid) or short (127 bp fragment) DNA molecules. The B-A transition of the fragment was found less cooperative than with linearized plasmid, which was confirmed by both CD spectroscopy and restriction cleavage inhibition.

Mapping the B-A conformational transition along plasmid DNA.

A simple method is presented to monitor conformational isomerizations along genomic DNA. We illustrate properties of the method with the B-A conformational transition induced by ethanol in linearized pUC19 plasmid DNA. At various ethanol concentrations, the DNA was irradiated with ultraviolet light, transferred to a restriction endonuclease buffer and the irradiated DNA was cleaved by 17 restriction endonucleases. The irradiation damaged DNA and the damage blocked the restrictase cleavage. The amount of uncleaved, i.e. damaged, DNA depended on the concentration of ethanol in a characteristic S-shape way typical of the cooperative B-A transition. The transition beginning and midpoint were determined for each restriction endonuclease. These data map the B-A transition along the whole polylinker of pUC19 DNA and six evenly distributed recognition sequences within the rest of the plasmid. The transition midpoints fell within the B-A transition region of the plasmid simultaneously determined by CD spectroscopy. The present method complements the previous methods used to study the B-A transition. It can be employed to analyze multikilobase regions of genomic DNA whose restriction endonuclease cleavage fragments can be separated and quantified on agarose gels.

Factors influencing resistance of UV-irradiated DNA to the restriction endonuclease cleavage.

DNA molecules of pUC19, pBR322 and PhiX174 were irradiated by various doses of UV light and the irradiated molecules were cleaved by about two dozen type II restrictases. The irradiation generally blocked the cleavage in a dose-dependent way. In accordance with previous studies, the (A + T)-richness and the (PyPy) dimer content of the restriction site belongs among the factors that on average, cause an increase in the resistance of UV damaged DNA to the restrictase cleavage. However, we observed strong effects of UV irradiation even with (G + C)-rich and (PyPy)-poor sites. In addition, sequences flanking the restriction site influenced the protection in some cases (e.g. HindIII), but not in others (e.g. SalI), whereas neoschizomer couples SmaI and AvaI, or SacI and Ecl136II, cleaved the UV-irradiated DNA similarly. Hence the intrastrand thymine dimers located in the recognition site are not the only photoproduct blocking the restrictases. UV irradiation of the A-form generally made the irradiated DNA less resistant to restrictase cleavage than irradiation in the B-form and in some cases, the A-form completely protected the UV-irradiated DNA against the damage recognized by the restrictases. The present results also demonstrate that the UV irradiation approach used to generate partial digests in genomic DNA studies, can be extended to the (G + C)-rich and (PyPy)-poor restriction sites. The present extensive and quantitative data can be used in genomic applications of UV damage probing by restrictases.