Kathleen Pietruszka Griffin

Absorption Spectrum of DNA for Wavelengths Greater than 300 nm

Although DNA absorption at wavelengths greater than 300 nm is much weaker than that at shorter wavelengths, this absorption seems to be responsible for much of the biological damage caused by solar radiation of wavelengths less than 320 nm. Accurate measurement of the absorption spectrum of DNA above 300 nm is complicated by turbidity characteristic of concentrated solutions of DNA. We have measured the absorption spectra of DNA from calf thymus, Clostridium perfringens, Escherichia coli, Micrococcus luteus, salmon testis, and human placenta using procedures which separate optical density due to true absorption from that due to turbidity. Above 300 nm, the relative absorption of DNA increases as a function of guanine-cytosine content, presumably because the absorption of guanine is much greater than the absorption of adenine at these wavelengths. This result suggests that the photophysical processes which follow absorption of a long-wavelength photon may, on the average, differ from those induced by shorter-wavelength photons. It may also explain the lower quantum yield for the killing of cells by wavelengths above 300 nm compared to that by shorter wavelengths.

P-Aminobenzoic acid can sensitize the formation of pyrimidine dimers in DNA: direct chemical evidence.

Thymine‐containing photoproducts with chromatographic properties similar to those of cyclobutyl pyrimidine dimers can be formed in [3H]‐thymine‐labeled DNA in solution by 313 nm ultraviolet radiation in the presence of para‐aminobenzoic acid (PABA), a compound used in sunscreen preparations. In the absence of PABA, similar fluences of 313 nm radiation do not produce significant numbers of these photoproducts. The thymine‐containing photoproducts can be reversed by 254 nm radiation so that the tritium label migrates with the mobility of thymine monomer, a behavior characteristic of thymine‐containing cyclobutyl pyrimidine dimers. This result supports previous, but less direct, data from other laboratories indicating that PABA can sensitize dimer formation in the DNA of bacterial and mammalian cells.

Simultaneous measurement of absorption and circular dichroism in a synchrotron spectrometer

Abstract We describe a method for simultaneously measuring the absorption spectrum and circular dicroism (CD) of a sample in a spectrometer that employs a transparent window photomultiplier detector. During the spectral scan, the computer control system simultaneously monitors the CD signal, the high voltage applied to the detector and the current in the storage ring. A servo-control system maintains the detector current at a constant value during the scan by varying the applied high voltage. The logarithm of the photomultiplier gain is calculated from the value of the high voltage using the predetermined characteristics of the tube. The gain value is corrected for the decrease of the storage ring current with time. During a single spectral scan, the computer generates a pseudo-absorption spectrum, which is the sum of the absorbance of the sample and of various wavelength-dependent system parameters. An absolute absorption spectrum is produced when the pseudo-absorption spectrum of a “blank” sample is subtracted from the desired sample spectrum. Absorption spectra measured by this method agree with spectra obtained with a double-beam spectrophotometer.

NETROPSIN: INTERACTION WITH ULTRAVIOLET IRRADIATED DNA

Abstract—Ultraviolet irradiation of double‐stranded DNA reduces the circular dichroism (i < 300 nm) induced when the basic peptide antibiotic netropsin (Nt) is added to DNA subsequent to thc irradiation compared to the CD induced by the same concentrations of Nt added to unirradiated DNA. Nt is known to bind to A T base pairs in duplex DNA but will not bind to single‐stranded DNA. The reduction in the maximum induced CD observed with saturating concentrations of Nt is a linear function of the concentration of pyrimidine dimers which. along with other dinucleotide photoproducts. form short disrupted regions in duplex DNA. The decrease in the CD of Nt bound to irradiated DNA could be due to elimination of potential Nt sites in the vicinity of a dimer. reduction in the average magnitude of the CD of Nt bound near a dimer or various combinations of these effects. In addition there is a reduction in the average binding constant for Nt bound to irradiated DNA compared to unirradiated DNA suggesting that formation of dinucleotide photoproducts either tends to preferentially eliminate the tighter binding sites or that tighter sites are converted to weaker ones. A simple model suggests that no more than one‐third to one‐half of the pyrimidine dimcrs formed in DNA completely eliminate a Nt site.

Photoreactivating enzyme from Escherichia coli: interactions with dna and mechanism of action

In photoreactivation, photochemical damage produced in DNA by far ultraviolet radiation (lambda < 320 nm) is repaired in an enzyme-mediated reaction using longer wavelength light (310 < lambda < 450 nm for E. coli). Photoreactivating enzyme (PRE) acts on a single class of photoproducts, cyclobutyl pyrimidine dimers, in an otherwise normal DNA strand at least nine bases long. PRE is one of the few DNA repair enzymes which has been purified to homogeneity in quantities sufficient for physico-chemical studies.