Top3 Processes Recombination Intermediates and Modulates Checkpoint Activity after DNA Damage

Abstract

Mutation of TOP3 in Saccharomyces cerevisiae causes poor growth, hyperrecombination, and a failure to fully activate DNA damage checkpoints in S phase. Here, we report that overexpression of a dominant-negative allele of TOP3, TOP3 Y356F , which lacks the catalytic (decatenation) activity of Top3, causes impaired S-phase progression and the persistence of abnormal DNA structures (X-shaped DNA molecules) after exposure to methylmethanesulfonate. The impaired S-phase progression is due to a persistent checkpoint-mediated cell cycle delay and can be overridden by addition of caffeine. Hence, the catalytic activity of Top3 is not required for DNA damage checkpoint activation, but it is required for normal S-phase progression after DNA damage. We also present evidence that the checkpoint-mediated cell cycle delay and persistence of X-shaped DNA molecules resulting from overexpression of TOP3 Y356F are downstream of Rad51 function. We propose that Top3 functions in S phase to both process homologous recombination intermediates and modulate checkpoint activity. INTRODUCTIONTopoisomerases are highly conserved proteins that catalyze topological rearrangements in the structure of DNA and are required for many aspects of DNA metabolism. Of the three topoisomerases in yeast, the function(s) of the sole type IA topoisomerase, Top3, remains most poorly understood. However, it is likely that Top3 fulfills important roles in vivo, because deletion of TOP3 in Saccharomyces cerevisiae causes hyperrecombination, sensitivity to genotoxic agents, meiotic defects, and poor growth due to accumulation of cells with a late S/G 2 content of DNA (Wallis et al., 1989;Gangloff et al., 1994Gangloff et al., , 1999Chakraverty et al., 2001). Similarly, deletion of top3 ϩ in Schizosaccharomyces pombe causes lethality due to chromosome missegregation and accumulation of DNA double-strand breaks (Goodwin et al., 1999;Maftahi et al., 1999;Oh et al., 2002;Win et al., 2004). Whereas lower eukaryotes generally contain only one type IA topoisomerase, human cells, like most vertebrates, possess at least two Top3 homologues, hTOPIII␣ and hTOPIII␤ (Hanai et al., 1996;Ng et al., 1999). In mice, mutation of TOP3␣ causes embryonic lethality (Li and Wang, 1998), whereas mutation of TOP3␤ causes a shortened life span (Kwan et al., 2003). Interestingly, in S. cerevisiae and S. pombe, deletion of SGS1 or rqh1 ϩ can largely suppress the phenotypes caused by deletion of TOP3 or top3 ϩ , respectively (Gangloff et al., 1994;Goodwin et al., 1999;Maftahi et al., 1999;Chakraverty et al., 2001). Because both SGS1 and rqh1 ϩ belong to the same family of proteins, the RecQ DNA helicases, this intriguing genetic interaction has led to the suggestion that type IA topoisomerases may be functionally associated with RecQ helicases. Indeed, both Sgs1 and Rqh1 physically interact with Top3 in their respective organisms (Gangloff et al., 1994;Bennett et al., 2000;Fricke et al., 2001;Onodera et al., 2002;Laursen et al., 2003;Ahmad and Stewart, 2005;Ui et al., 2005), and a close as...

Shu Proteins Promote the Formation of Homologous Recombination Intermediates That Are Processed by Sgs1-Rmi1-Top3

“…This prolongation of S-phase by MMS (relative to unperturbed cells) is a consequence of MMS-induced replication fork stalling and subsequent checkpoint activation (Paulovich and Hartwell, 1995;Santocanale and Diffley, 1998;Shirahige et al, 1998;Tercero and Diffley, 2001). In agreement with our previous data (Mankouri and Hickson, 2006), rad51 cells failed to completely traverse S-phase by 4 h in the presence of 0.0167% MMS, demonstrating a mid-Sphase DNA content at 4 h ( Figure 2B). Interestingly, we observed that rad54 and shu1 mutants resembled rad51 mutants and also failed to traverse S-phase by 4 h in 0.0167% MMS ( Figure 2B).…”

“…Plates were grown at 30°C. The large single colonies that arise in strains overexpressing TOP3 Y356F represent suppressors (probably SGS1-linked) as seen previously (Oakley et al, 2002;Mankouri and Hickson, 2006). (B) Wild-type, shu1, rad51, rad51shu1, rad54, and rad54shu1 strains were diluted to equivalent densities and spotted onto control plates (no drug) or plates containing MMS.…”

“…All haploid rmi1 strains used for our experimental analyses were confirmed to demonstrate a slow-growth phenotype (Chang et al, 2005;Mullen et al, 2005). The pYES2-TOP3 and pYES2-TOP3 Y356F plasmids have been described previously (Oakley et al, 2002;Mankouri and Hickson, 2006). …”

“…Release from MMS treatment was achieved by centrifugation, washing, and resuspension of cells in drug-free medium. Cell cycle progression was monitored using flow cytometry (fluorescent-activated cell-sorting [FACS]) as described previously (Mankouri and Hickson, 2006). …”

Genetic evidence for a role of Saccharomyces cerevisiae Mph1 in recombinational DNA repair under replicative stress

“…In particular, Sgs1 and Top3 are suggested to resolve recombination structures originating from sister chromatid junctions at damaged replication forks, while Srs2 prevents their formation (Liberi et al, 2005;Mankouri and Hickson, 2006;Mankouri et al, 2007). mph1 mutants are not deficient in HR, as determination of spontaneous mitotic recombination rates demonstrated.…”