SUMMARYA re-analysis of the metabolic fate of ultraviolet light (u.v.)-induced cyclobutyl pyrimidine dimers in the DNA of dermal fibroblasts from patients with different genetic forms of xeroderma pigmentosum (XP), a rare cancer-prone skin disorder, has provided new insight into the mode of dimer repair in normal human cells. When DNA isolated from post-u.v. incubated cultures was subjected to enzymic photoreactivation (PR) to probe dimer authenticity, single-strand scissions were produced in the damaged DNA of incubated XP group A and D cells, but not in DNA from XP group C cells or normal controls. Since enzymic PR treatment ruptures only the cyclobutane ring, these results suggested that in dimer excision-defective XP group A and D strains, the intradimer phosphodiester bond may have been cleaved without site restoration. Such a cleavage event had not previously been detected; the possibility that this reaction may be an early step in the normal excision-repair process is supported by the observed release of free thymidine (dThd) and its monophosphate (TMP), but not of thymine, upon photochemical reversal of the dimercontaining excision fragments isolated from post-u.v. incubated normal cells. The combined number of dThd and TMP molecules released was equal to =80% of the number of dimers photoreversed; for such release to occur, the dimer must both be at one end of an excised fragment and contain an internal phosphodiester break. Taken together, these data lead us to propose a novel model for dimer repair in human cells in which hydrolysis of the intradimer phosphodiester linkage precedes the concerted action of a generalized 'bulky lesion-repair complex' involving conventional strand incision/lesion excision/repair resynthesis/strand ligation reactions.
A non-transformed human fibroblast strain, GM11, established from the skin of a therapeutically aborted fetus, has been reported to exhibit the Mer- phenotype, i.e. inability to support the growth of adenovirus 5 damaged with 1-methyl-3-nitro-1-nitrosoguanidine. In the present study we determined (i) loss of colony-forming ability and frequency of mutants resistant to 6-thioguanine (6TG) on exposure to the SN1 alkylating agent methylnitrosourea (MNU) and (ii) amount of O6-methylguanine-DNA methyltransferase (MGMT), the protein responsible for repairing O6-methylguanine (O6mG) produced by MNU, in GM11 cells compared to GM10, a Mer+ human fetal fibroblast strain. Irrespective of in vitro culture age, GM10 cells responded normally to the cytotoxic action of the alkylating agent, i.e. their clonogenic survival curves exhibited a shoulder at low MNU concentrations (less than or equal to 0.4 mM) and a D10 (dose reducing survival to 10%) of approximately 1.4 mM. By contrast, no shoulder was observed on the survival curves of GM11 cells and their D10 values decreased from approximately 0.6 mM at passage 4 to 0.1 mM at passage 27. In GM10 (Mer+) cells, unlike the biphasic dose response seen for cell killing, the frequency of 6TG-resistant mutants increased as a linear function of chemical concentration delivered (range 0.05-1.2 mM); the induced mutation frequency in these cells (passage 16-20) was equal to 220 x 10(-6)/mM MNU, a yield some 5-fold greater than that reported by others for non-fetal human fibroblasts. GM11 cells proved to be only approximately 1.5 times more mutable by MNU than GM10 cells at late passage, and the susceptibility of the former strain to MNU-induced mutations did not change significantly as a function of culture age (i.e. 316 x 10(-6) and 326 x 10(-6) mutants/mM MNU at passages 4 and 16-20 respectively). The GM10 strain contained approximately 75,000 MGMT molecules/cell at all passages (4-20) examined, whereas the GM11 strain harbored deficient amounts of the protein (approximately 22,500 molecules/cell) at the lowest passage available (4), and this residual activity decreased precipituously to undetectable amounts by passage 16. Together, these data demonstrate that in the two human fetal strains examined the constitutive level of cellular MGMT activity correlates much better with resistance to reproductive inactivation than with mutagenesis by MNU, implying that inefficient repair of O6meG lesions impacts more severely on cell lethality than on mutation induction in at least some biological systems.
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