The Influence of DNA Configuration on the Direct Strand Break Yield.

PURPOSE: To study the influence of DNA configuration on the direct damage yield. No indirect effect has been accounted for. METHODS: The GEANT4-DNA code was used to simulate the interactions of protons and alpha particles with geometrical models of the A-, B-, and Z-DNA configurations. The direct total, single, and double strand break yields and site-hit probabilities were determined. Certain features of the energy deposition process were also studied. RESULTS: A slight increase of the site-hit probability as a function of the incident particle linear energy transfer was found for each DNA configuration. Each DNA form presents a well-defined site-hit probability, independently of the particle linear energy transfer. Approximately 70% of the inelastic collisions and ~60% of the absorbed dose are due to secondary electrons. These fractions are slightly higher for protons than for alpha particles at the same incident energy. CONCLUSIONS: The total direct strand break yield for a given DNA form depends weakly on DNA conformation topology. This yield is practically determined by the target volume of the DNA configuration. However, the double strand break yield increases with the packing ratio of the DNA double helix; thus, it depends on the DNA conformation.

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.