The response of mammalian cells to alkylating agents: II. On the mechanism of the removal of sulfur-mustard-induced cross-links

Abstract

1. The ability of L-cells to remove alkylation products produced by sulfur mustard di-(2-chloroethyl)-sulfide from their DNA has been studied. 2. The loss of total alkylation products as well as the loss of the diguaninyl mustard product, di-(guanin-7-yl)-ethyl sulfide, were followed as a function of time using 35S-labelled mustard. Both processes were exponential and occurred with half-lives of 18 h. 3. Parallel measurements of the amount of renaturable cross-linked DNA present were made using methylated albumin kieselguhr column chromatography or ultracentrifugation in CsCl gradients to separate renaturable, i.e., bihelical, DNA from non-renaturable, i.e., denatured, DNA. These studies showed that (a) the loss of renaturable DNA occurred with a half-life of 2 h; (b) the loss of renaturable DNA occurred more rapidly than did the loss of either the total or the diguaninyl alkylation products; (c) this loss of renaturable DNA occurred in the absence of preferential excision of the diguaninyl alkylation product. 4. The following mechanism is proposed to account for these findings. It appears that L-cells are able to excise both monoguaninyl (7-hydroxyethylthioethylguanine) and diguaninyl alkylation products from their DNA and further, that the latter product is removed by a two-step process that involves first, the ‘unhooking’ of one arm of the cross-link and second, excision at some later time of the remaining product.