10][“Metabolism R”

Hence, the excision of an oligomer carrying the phosphorothioate bond was unexpected and led us to consider the possibility that the excision nuclease system, which is capable of recognizing such subtle perturbations in the duplex structure, may act in a similar manner on unmodified DNA with a low but finite probability.When we tested the unmodified DNA in our assay system, we found that fragments nucleotides in length were removed from this substrate as well. That these oligomers are generated by the excision nuclease system and not by nonspecific degradation of DNA by contaminating nucleases is supported by three lines of evidence.Finally, extracts from cells lacking the XPG or XPF subunits of the excinuclease fail to release mers from undamaged DNA.Moreover, the excision activity can be restored by supplementing extract from the mutant cell line with the missing subunit, considered in its entirety, show that the human excision nuclease is capable of excising oligomers of nt nominal length from undamaged DNA.Similar levels of excision were observed when the centrally located oligomer with P label in the bp duplex was an undamaged mer TCCTCCTCGCCTCCT or mer, GCTCGAGCTAAATTCGTCAG. Thus, it appears that excision of undamaged DNA occurs in at least three different sequence contexts.Substrate DNA prepared with unmodified oligonucleotides was incubated for min in ml reaction mixtures either lacking or containing cellfree extracts prepared from normal cells, or XPF extract supplemented with purified protein or with extract prepared from an XPG cell line.The figure shows an autoradiograph obtained after resolution of DNA samples in a polyacrylamide sequencing gel; brackets indicate the location of excision products.Hence, it could be argued that the excision we observe from our nominally undamaged DNA may be due to low levels of oxidative damage introduced during the handling of DNA.To address this concern we measured the level of oxoG in our synthetic oligonucleotides that had been subjected to essentially the same treatment as our radiolabeled substrate.OxoG is the most common oxidative stress lesion and, among the major oxidative base lesions, it is the most efficient substrate for human excision nuclease. Thus, we reasoned that if excision from undamaged DNA arises from cryptic lesions, most of it would have been caused by oxoG.The rate of excision from undamaged DNA is the rate of removal of a single oxoG in the same duplex. It should be noted, however, that our quantitation of oxoG was performed with nonradiolabeled DNA.An argument could therefore be made that with radiolabeled DNA the oxoG level would be greater due to DNA damage caused by radioactive decay, and thus there would be higher contributions to the excision signal from damage.However, we think this is unlikely to be the source of gratuitous excision for the following reason.The DNA molecules in which the decay occurs are no longer detectable in the excision assay, and the likelihood that low level bdecay would damage other DNA molecules, especially in the presence of EDTA in the storage buffer, is infinitesimally small.Thus, it can be reasonably concluded that most of the excision signal we detect with undamaged DNA is produced by the attack of the excision nuclease on undamaged DNA as a consequence of the intrinsic property of the action mechanism of the excision nuclease system.

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