Multiple endonucleases function to repair covalent topoisomerase I complexes in Saccharomyces cerevisiae

Abstract

Topoisomerase I plays a vital role in relieving tension on DNA strands generated during replication. However if trapped by camptothecin or other DNA damage, topoisomerase protein complexes may stall replication forks producing DNA double-strand breaks (DSBs). Previous work has demonstrated that two structure-specific nucleases, Rad1 and Mus81, protect cells from camptothecin toxicity. In this study, we used a yeast deletion pool to identify genes that are important for growth in the presence of camptothecin. In addition to genes involved in DSB repair and recombination, we identified four genes with known or implicated nuclease activity, SLX1, SLX4, SAE2, and RAD27, that were also important for protection against camptothecin. Genetic analysis revealed that the flap endonucleases Slx4 and Sae2 represent new pathways parallel to Tdp1, Rad1, and Mus81 that protect cells from camptothecin toxicity. We show further that the function of Sae2 is likely due to its interaction with the endonuclease Mre11 and that the latter acts on an independent branch to repair camptothecin-induced damage. These results suggest that Mre11 (with Sae2) and Slx4 represent two new structure-specific endonucleases that protect cells from trapped topoisomerase by removing topoisomerase-DNA adducts.

Figure 1.—

Loss of Slx4 but not Slx1 sensitizes tdp1mutants to camptothecin. (a) Isogenic wild-type, tdp1, slx4, and slx4 tdp1 single- and double-deletion strains were exposed to camptothecin at various concentrations. Their sensitivities were assessed by the ratio of slope of the growth curve of the treated (c) vs. the untreated (c = 0) (see materials and methods). (b) Isogenic wild-type, tdp1, slx1, and slx1 tdp1 single- and double-deletion strains were tested as in a.

Figure 2.—

Epistasis study of slx4, rad1, and mus81. Experiments were carried out as in Figure 1. (a) The slx4 rad1 tdp1 triple-deletion strain is more sensitive to camptothecin than either the slx4 tdp1 or the rad1 tdp1double-deletion strain. (b) The slx4 rad1 double-deletion strain is not more sensitive to camptothecin than either single-deletion strain. (c) The slx4 mus81 tdp1 triple-deletion strain is more sensitive than the slx4 tdp1 and mus81 tdp1 double-deletion strains. Standard errors for three replicate experiments are shown but are smaller than the points.

Figure 3.—

Sae2 is required for protection from camptothecin toxicity. (a) Deletion of SAE2 caused substantial sensitivity compared with wild type or the tdp1 strain. Deletion of TDP1 in the sae2 strain caused slight additional sensitivity. (b) The tdp1 rad1 sae2 triple-deletion strain is more sensitive than either the tdp1 rad1 or the tdp1 sae2 double-deletion strain. (c) Similarly, the tdp1 sae2 mus81 triple-deletion strain showed more sensitivity than the double-deletion strain of tdp1 sae2 or tdp1 mus81.

Figure 4.—

Mre11 endonuclease domain is required for protection from camptothecin, and its function is in the same epistasis group as Sae2. (a) The mre11-H125N mutation strain caused mild sensitivity to camptothecin, which is increased by further deletion of TDP1. The triple-deletion strain mre11-H125N tdp1 rad1 is more sensitive than either of the double-deletion strains tdp1 mre11-H125N or tdp1 rad1. (b) The sae2 mre11-H125N double-mutant strain shows essentially the same sensitivity as the sae2 deletion strain, indicating that they are in the same epistatic pathway.

Figure 5.—

Sae2 acts on Top1 lesions and is epistatic with Mre11 and not with Rad9. (a) The sensitivity of the sae2 deletion strain to camptothecin is abrogated by deletion of TOP1, showing that Sae2 is acting on lesions produced by Top1. (b) The sae2 mre11 double-deletion mutant is no more sensitive than the mre11 deletion strain, demonstrating that the two operate in the same repair pathway. Also shown are three replicate experiments (with standard errors) for a rad52 deletion strain. (c) Loss of Sae2 and Rad9 produces additive sensitization to camptothecin.

Figure 6.—

Diagram of pathways repairing camptothecin-stabilized Top1 complex. Various lines of evidence indicate that the substrate for repair is a double-strand break arising from collision of a replication fork with the covalently attached Top1 (Pouliot et al. 1999). (Top) Spontaneous cutting and religation by Top1 can occur, although at a slower rate because camptothecin inhibits the ligation step. Stalled replication forks can then be restarted possibly by Mus81. (Middle) Alternatively, Tdp1 can resect Top1 off the DNA backbone, leaving a phosphate group at the 3′ end of DNA. The phosphate group can then be removed by the redundant phosphatases Apn1, Apn2, and Tpp1. (Bottom) Several endonucleases can directly remove the covalently bound Top1 together with a DNA fragment. These endonucleases have different site selectivities, implying that different DNA intermediates exist with stalling of replication forks caused by camptothecin.