Mehrnaz Katouzian-Safadi’s research while affiliated with French National Centre for Scientific Research and other places

This study compares the photo- and radiosensitizing effect of 5-bromouracil (BrU) on breakage of double stranded DNA. Upon U.V. irradiation, we observe a hot spot of breakage at the nucleotide in 5' from the BrU. Upon X irradiation, the breakage probability at the same site is only slightly enhanced. This result shows that the radiosensitizing effect of BrU in cell inactivation cannot be explained by an overproduction of strand breaks.

Photochemical induced cross-links between protein and nucleic acids are useful tools in the study of the protein-DNA interactions. The substitution of thymine by 5-bromouracil in DNA increases the photocross-linking yield, and reduces the direct damages to both DNA and proteins. Using the lac repressor-DNA non-specific interaction system, we have developed a procedure to identify the interaction site on the protein. Sensitive, accurate and inexpensive in time and material, this procedure is based on the possibility of sequencing peptides in the presence of a large excess of DNA. The obtained result (the implication of His 29) agrees with previous work.

Adenosine cyclic 3',5'-phosphate receptor protein (CRP or CAP) is a regulatory protein involved in the transcription of several operons in Escherichia coli. cAMP-independent, nonspecific complexes of CRP and DNA were investigated by photochemical cross-linking of the protein to nonspecific DNA, whose thymines are substituted by 5-bromouracil (BrUra). The cross-linked protein was completely digested by trypsin, and the covalently bound peptides were sequenced. We identified two regions of the protein in close contact with DNA: one in the C-terminal part, overlapping the canonical helix-turn-helix motif, and the other one in the N-terminal part, which is usually not considered to belong to the DNA-interacting domain of CRP. This result lead us to propose models for nonspecific interaction, where the DNA is in contact with both the N- and C-terminal parts of the protein.

Protein MC1 is the major chromosomal protein in methanosarcinaceae. Using photochemical crosslinking on 5-bromouracil-substituted DNA, we identified the region of the protein that interacts with it. This region is located in the C-terminal part of the polypeptlde chain, and the crosslinked amino-aclds are in the region 74–86. Tryptophan 74 is one of the aminoacids crosslinked to DNA.

Cyclic AMP receptor protein (CRP) is a regulatory protein implicated in the transcription of several operons in Escherichia coli. Its activity is modulated by effectors, such as cAMP or cGMP, which could induce (or not) structural changes in the protein, and activate (or not) the transcription. CRP can bind non-specifically to DNA, and we investigated the photocross-linking of the protein to E. coli 5-bromouracil-substituted DNA, in the absence and in the presence of effectors. The photochemistry of the protein alone, under the conditions used for the cross-linking reaction, was studied. We show that tryptophyl residues are more photoreactive than tyrosyl ones. Photocross-linking of the protein implicates only one of the two subunits, and the rate of the reaction is not modified upon cAMP binding. Binding of cGMP reduces the rate of photocross-linking by a factor of two. These new results show that the protein in the CRP-cGMP complex behaves differently from the free protein.

Abstract— Cyclic AMP receptor protein (CRP) is a regulatory protein implicated in the transcription of several operons in Escherichia coli. Its activity is modulated by effectors, such as cAMP or cGMP, which could induce (or not) structural changes in the protein, and activate (or not) the transcription. CRP can bind non-specifically to DNA, and we investigated the photocross-linking of the protein to E. coli 5-bromouracil-substituted DNA, in the absence and in the presence of effectors. The photochemistry of the protein alone, under the conditions used for the cross-linking reaction, was studied. We show that tryptophyl residues are more photoreactive than tyrosyl ones. Photocross-linking of the protein implicates only one of the two subunits, and the rate of the reaction is not modified upon cAMP binding. Binding of cGMP reduces the rate of photocross-linking by a factor of two. These new results show that the protein in the CRP-cGMP complex behaves differently from the free protein.

4',6-Diamidino-2-phenylindole, DAPI, forms fluorescent complexes with DNA. This property has been used to quantify DNA on the basis of fluorometric test. However, the fluorescence quantum yield of DAPI increases also with tRNA. DNA estimation needs particular care in the presence of tRNA. For DNA containing 50% adenine-thymine (AT), DAPI can be used if DNA represents at least 3.4% of the total nucleotide material. This percentage varies with the AT/guanosine-cytosine content. When the fraction of DNA decreases further, the DAPI assay can no longer be used.

We examined the circular dichroism spectra of intact Turnip yellow mosaic virus, freezed/thawed virus, empty capsid particles, and phenol extracted RNA. The circular dichroism signal of the empty capsid was found to contribute for less than 1% to the circular dichroism of the virus. Differences in the circular dichroism spectra indicate that TYMV-RNA exhibits different conformations when it is in situ in the virus, when it has been ejected by freezing/thawing and when it has been phenol extracted. Increase of the ionic strength up to 0.1 M NaCl led to conformational change of the RNA either freezed/thawed ejected or phenol extracted but not in situ in the capsid. Addition of spermidine (3 mM) induced a conformational change only for the phenol extracted RNA. These results are discussed with respect to the origin of the various conformational states of viral RNA.

Turnip yellow mosaic virus (TYMV) RNA escapes from viral capsids after freezing and thawing the virus, and the remaining capsids look very similar to natural capsids in the electron microscope after negative staining [Katouzian-Safadi, M., Favre, A., and Haenni, A. L. (1980) Eur. J. Biochem. 112, 478-486]. In order to understand how an RNA of 2 X 10(6) Da (33% virus by weight) can escape from a compact protein shell we have compared artificial capsids formed after freezing TYMV and natural capsids produced in vivo in infected plants. We have used various physicochemical techniques including analytical ultracentrifugation, X-ray scattering, X-ray diffraction and orientation in a magnetic field. From the combination of these results we conclude that the escape of the RNA is accompanied by the formation of a hole in the capsid surface. The size of such a hole is estimated to 5-9 coat protein subunits.