We have investigated the levels of nuclear protein acetylation in u.v.-irradiated human fibroblasts. Initially, we measured the levels of acetylation in total acid-soluble nuclear proteins and observed two distinct differences between the irradiated and unirradiated (control) cells. Immediately after irradiation, there is a 'wave' of protein hyperacetylation (i.e. a total acetylation level greater than that of unirradiated cells) that lasts for 2-6 h depending on the experimental conditions. This hyperacetylation phase is then followed by a hypoacetylation phase, lasting for many hours, and the total level of acetylation does not return to that of control cells until 24-72 h after u.v. damage. Both the magnitude and duration of each phase is dependent on the dose of u.v. light used. The wave of hyperacetylation is more pronounced at low u.v. doses (i.e. less than 5 J/m2), while the wave of hypoacetylation is more pronounced at higher u.v. doses (greater than or equal to 8 J/m2). Furthermore, the duration of each phase is prolonged when cells are exposed to 2 mM hydroxyurea, an agent which retards the rate of excision repair at u.v.-damaged sites. Examination of the acetylation levels of the individual nuclear proteins indicated that acetylation of the core histones follows the same pattern observed for the total acid-soluble protein fractions. Furthermore, these were the only major proteins in the total acid-soluble fraction observed to undergo the early, rapid hyperacetylation immediately following u.v. damage. Acetylation of histone H1 was negligible in both damaged and control cells, while three prominent non-histone proteins were acetylated only after long labeling times (greater than 4 h) in each case, gradually becoming hyperacetylated in the u.v.-damaged cells. These results raise the possibility that a causal relationship exists between nuclear protein acetylation and nucleotide excision repair of DNA in human cells.

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SynopsisBinding isotherms can provide quantitative information regarding the stability of a molecular complex. Theoretical studies in recent years have been directed to systems in which a single ligand can exclude more than one polymer site (excluded-site effect). This system has a minimum of three parameters to describe the binding data: the intrinsic binding constant, B; the remote-neighbor cooperativity parameter, uq; and the number of excluded sites, q. It is suggested in the present communication that precise values for these three parameters can be obtained by utilizing the characteristics of two forms of data representation: 0 vs In m and 8/m vs 0, where 0 is the degree of saturation (0 5 0 5 1) and m is the molality of free ligand. The matrix generation method is used to obtain empirical equations relating the midpoint location and slope at the midpoint of the 0 vs In m plot to the three molecular parameters. A modified Scatchard theory is also presented for highly cooperative systems, which results in an expression relating the maximum in the elm vs 0 plot to the molecular parameters uq and q , thus providing the third equation for the three unknown parameters. The novel method of analysis is illustrated with the AMP-poly(L-arginine) and oligocytidylate-T7 DNA systems.

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