Repair of intermediate structures produced at DNA interstrand cross-links in Saccharomyces cerevisiae

Abstract

Bifunctional alkylating agents and other drugs which produce DNA interstrand cross-links (ICLs) are among the most effective antitumor agents in clinical use. In contrast to agents which produce bulky adducts on only one strand of the DNA, the cellular mechanisms which act to eliminate DNA ICLs are still poorly understood, although nucleotide excision repair is known to play a crucial role in an early repair step. Using haploid Saccharomyces cerevisiae strains disrupted for genes central to the recombination, nonhomologous end-joining (NHEJ), and mutagenesis pathways, all these activities were found to be involved in the repair of nitrogen mustard (mechlorethamine)- and cisplatin-induced DNA ICLs, but the particular pathway employed is cell cycle dependent. Examination of whole chromosomes from treated cells using contour-clamped homogenous electric field electrophoresis revealed the intermediate in the repair of ICLs in dividing cells, which are mostly in S phase, to be double-strand breaks (DSBs). The origin of these breaks is not clear since they were still efficiently induced in nucleotide excision and base excision repair-deficient, mismatch repair-defective, rad27 and mre11 disruptant strains. In replicating cells, RAD52-dependent recombination and NHEJ both act to repair the DSBs. In contrast, few DSBs were observed in quiescent cells, and recombination therefore seems dispensable for repair. The activity of the Rev3 protein (DNA polymerase zeta) is apparently more important for the processing of intermediates in stationary-phase cells, since rev3 disruptants were more sensitive in this phase than in the exponential growth phase.

FIG. 1

HN2 sensitivity of the rad52 strain (WXY9387) and its isogenic parent (DBY747) in the exponential and stationary growth phases. (A) Exponentially growing cells were treated with the stated doses for 1 h at 28°C. Appropriate dilutions giving around 200 colonies on untreated controls were spread on YEPD plates and incubated for 3 days. Also shown for reference are the results obtained with an isogenic rad14 disruptant. (B) As in panel A but using stationary-phase cells. All results are the means of at least three independent experiments, and the vertical error bars show the standard error of the mean.

FIG. 2

Sensitivity of parental (W303-1B), rad52, yku70, and yku70 rad52 strains to HN2 and the monofunctional mustard HN1. (A) Exponential-phase cells were treated with 0 to 1,000 μM HN2. (B) Stationary-phase cells were treated with 0 to 1,000 μM HN2. (C) Exponential-phase cells were treated with doses from 0 to 10,000 μM HN1. All results are the means of at least three independent experiments, and the vertical error bars show the standard error of the mean.

FIG. 3

The sensitivity of parental (W303-1B), rad52, yku70, and yku70 rad52 strains to cisplatin also depends on growth phase. Exponential-phase cells (A) and stationary-phase cells (B) were treated with 0 to 1,000 μM cisplatin, and survival was monitored as described in Materials and Methods. All results are the means of at least three independent experiments, and the vertical error bars show the standard error of the mean.

FIG. 4

Survival following HN2 treatment of parental and rev3 cells with HN2 in the exponential and stationary growth phases. DBY747 and rev3 cells from exponential- or stationary-phase cultures were treated with HN2 at the doses shown, and survival was determined as described in Materials and Methods.

FIG. 5

Induction of DSBs in exponential-phase (Exp DBY) and stationary-phase (Stat DBY) parental (DBY747) cells and rad52 exponential-phase (Exp rad52) cells determined by CHEF. Cells were treated with 0, 10, 100, and 1,000 μM HN2 for 3 h and subjected to a 1-h posttreatment incubation to allow time for incision. The cells were embedded in agarose, and chromosome preparations were run on CHEF gels as described in Materials and Methods. The position of the well (w) is marked on the gel.

FIG. 6

Induction of DSBs following HN2 (0, 100, and 1,000 μM) treatment of exponentially growing DBY747 and isogenic rad4, rad4 mag1, rad2, and rad1 rad2 disruptants determined by CHEF.

FIG. 7

Repair of DSBs in HN2-treated W303-1B, rad52, yku70, and yku70 rad52 cells. (A) Exponentially growing cells were treated with 100 μM HN2 or mock treated (lanes U) with water and subsequently allowed to repair in minimal medium for 2, 4, or 24 h. The mock-treated sample was allowed to repair for 24 h. The samples were analyzed on CHEF gels. (B) Exponentially growing rad52 cells derived from DBY747 were treated and analyzed by CHEF in an identical manner to those in panel A.