Protein--nucleic acid interactions are of key importance in gene regulation. The laser strategy for studying these interactions is based on the rapid "freezing" of the processes through specific, single pulse-induced, irreversible photoreactions. Two different approaches, either protein--DNA crosslinking or footprinting, may be used, depending on the object to be studied. Protein--DNA crosslinking detects the presence of a protein on a given DNA sequence, whereas footprinting sheds light on the mechanism and details of protein--DNA interactions.
Laser Protein--DNA Photofootprinting
UV irradiation of protein--DNA complexes results in different types of lesions in DNA, the lesion spectrum depending on the presence and type of proteins in these complexes (1). This dependence is determined by local conformational changes in DNA induced by protein--DNA interactions. In fact, UV light "feels" local DNA structure. Thus, it can be used as a probing agent for analysis of both protein--DNA interactions and DNA conformation. The method developed for this analysis is called "photofootprinting". The use of UV lasers has many advantages compared to conventional light sources. With a single UV laser pulse, a footprint of the protein is achieved. Additionally, high-intensity laser irradiation, contrary to conventional light sources, induces specific biphotonic lesions in DNA. These lesions are extremely sensitive to local DNA structure and can be easily detected by cleavage with chemical reagents or enzymatic digestion.
Two well-studied, laser-specific guanine lesions are 8-oxodG and oxazolone (2,3). 8-oxodG is quantitatively cleaved by Fpg protein, whereas oxazolone is removed upon hot piperidine treatment. Separation of the cleaved, irradiated DNA allows us to find the positions of the lesions. The chemical mechanism of formation of these two types of lesions is well documented. These lesions originate from the same type of initial radical cations and are induced by a competitive transformation process; hydration at position 8 of guanine radical cation leads to the formation of 8-oxodG, whereas oxazalone is formed upon radical cation deprotonation. The sum of quantum yields of both lesions is equal to the guanine photoionization quantum yield (4). Thus, the cleavage of irradiated protein--DNA samples with these two reagents enables us to judge the distortion of DNA structure induced by the bound protein. For example, if the presence of a protein on a DNA sequence decreases the accessibility of water molecules to a guanine base, this will result in a diminution of the relative yield of 8-oxodG, whereas the weakening of G-C pairing will lead to an enhancement of oxazolone quantum yield (4).
The described UV laser photofootprinting technique is a novel approach, and its first application on histone H1°-four-way-junction DNA (4WJ DNA) was recently described in the literature (5). It was found that the binding of histone H1° affects the photoreactivity of some guanine residues located on the central part of the four-junction DNA. It should be noted that the other existing, high-resolution footprinting techniques enabled us to detect such H1°-4WJ DNA interaction. This demonstrates the higher sensibility of this novel UV laser footprinting technique.
UV Laser-induced Protein--DNA Crosslinking
The UV laser crosslinking, as in the case of laser protein--DNA footprinting, exhibits several advantages over techniques using low-intensity light sources. These advantages are determined by the high amount of photons delivered by the laser in a single-nanosecond or -picosecond pulse. This high photon concentration allows the protein--DNA crosslinking reaction to operate via a biphotonic mechanism through the intermediary of radical cations of nucleic bases 6, 7, 6, 1. The laser-induced generation of radical cations exhibits a high quantum yield, which leads to a higher efficiency of crosslinking (exceeding by close to two orders of magnitude that obtained with conventional UV light sources). This makes possible the formation of crosslinks that cannot be induced by conventional UV lamps (6). The crosslinking reaction itself is completed in much less than 1 μs, which avoids the possibility of artifactual crosslinking of UV-damaged molecules and permits the trapping of rapid dynamic changes in protein--DNA interactions (7).
UV laser crosslinking has been used in vitro for studying different aspects of protein--DNA interactions including measurements of binding constants, the determination of protein--DNA contact points, the size of the protein--nucleic acid complexation site [reviewed by (7)], etc. The technique can provide information that is not accessible via other approaches. For example, upon irradiation of nucleosomes with UV laser, the histone--DNA crosslinking was found to occur only via the nonstructural histone NH2 tails and thus presented a unique tool for studying these histone domain interactions with DNA (8). This finding has allowed us to demonstrate that the tails are not released from nucleosomal DNA upon histone acetylation, a question that has been discussed for many years in the literature (9). In addition, the UV laser crosslinking method has permitted the transient DNA--ATPase complex in the T4 DNA replication system to be "frozen" and, hence, the dynamics of protein--DNA interactions to be investigated (7). The technique was also successfully applied in kinetic studies on the interaction of Escherichia coli RNA polymerase with the promoter DNA sequence (10) as well as to the TBP (TATA-box binding protein) assembly with the adenovirus E4 promoter (11).
A procedure for in vivo UV laser crosslinking of chromosomal proteins to DNA was also developed (11). This procedure, in combination with immunochemical techniques, is used to detect in vivo the presence of specific proteins on DNA sequences of interest [for details, see (12)].
Conclusions and Perspectives
The few examples described in this brief communication demonstrate an important property of laser protein--DNA crosslinking and footprinting approaches---the possibility to "freeze", by a single nanosecond or picosecond pulse, labile protein--DNA complexes that can be analyzed further by standard molecular biology techniques. This property of the laser methods could be used in both in vitro and in vivo studies. For example, synchronization of a laser pulse with a stop-flow technique will prompt investigations of rapid protein--DNA interactions, which, in a combination with a procedure for mapping protein--DNA contact points, could be successfully applied in dynamic protein--DNA recognition studies.
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