Our data suggest that SUMO attachment to Flp recombinase prevents Flp from causing DNA damage. We propose that when Flp is not sumoylated, Flp-dependent damage is repaired by the cellular HRR pathway, ultimately leading to formation of a HMW species containing multiple tandem copies of the 2 μm sequence. This aberrant process is associated with the elevated levels of 2 μm DNA in SUMO pathway mutants. There is no reason to think that normal Flp-dependent 2 μm amplification in wt cells occurs by the same mechanism. The SUMO pathway could also affect 2 μm behavior via additional mechanisms.
The effect of SUMO on Flp appears to be mediated by Slx5·Slx8. The most likely explanation for this is that Slx5·Slx8 targets sumoylated Flp for ubiquitin-dependent proteolysis. However, we have not been able to detect a difference in the levels of Flp ubiquitylation or degradation between wt and siz1Δ siz2Δ or slx5Δ or slx8Δ mutants, using overexpressed Flp (not shown). One explanation for this would be if Flp can be ubiquitylated and degraded via multiple pathways, where SUMO-dependent degradation accounts for a relatively small fraction, at least when Flp is overexpressed. For example, SUMO- and Slx5·Slx8-dependent ubiquitylation might target only DNA-bound Flp. This model would explain the result that Flp causes DNA damage in SUMO pathway mutants, because increased residence of Flp at the DNA would increase the chance that the Flp-DNA covalent intermediate would be encountered by a RF and converted to a double-strand break. This model would also explain why overexpression of Flp in wt cells did not have the same effect as reducing Flp sumoylation (), because overexpressed Flp could still be removed from the DNA by the SUMO pathway.
Several observations suggest that amplification of 2 μm in SUMO pathway mutants may occur via BIR (, , and ). However, BIR mutants such as rad51
Δ had only an approximately twofold effect on levels of the HMW species, indicating that the two known BIR pathways (RAD51
-dependent and RAD51
-independent) are not absolutely required for formation of the HMW species. This could suggest 1) that BIR is occurring through a third mechanism, 2) that the requirements for BIR are relaxed when 2 μm is the template, or 3) that the HMW species can form by a mechanism other than BIR. rad51
Δ and rad54
Δ eliminate most, but not all, BIR (McEachern and Haber, 2006
; Llorente et al., 2008
), indicating that there are BIR mechanisms that do not require these pairs of genes. Furthermore, the presence of a circular template might reduce the need for some features of the BIR mechanism. There are several models for how BIR takes place, some of which involve formation of a Holliday junction (HJ) behind the RF (McEachern and Haber, 2006
; Llorente et al., 2008
). This HJ could either be resolved or could branch-migrate behind the RF. BIR on a circular template would not require either of these mechanisms to deal with the HJ, because the RF could travel around the template until it came up behind the HJ, where it might “push” branch migration of the HJ (E). A non-BIR mechanism could also produce the structure in E: during plasmid replication, one RF could stall at Flp-bound FRT and regress, forming a “chicken foot” HJ-like structure. If the fork from the other side of the plasmid then passed the FRT site, it might push this regressed fork/HJ backward, extruding a linear tail, where the end represents the site where the fork initially stalled. If the HMW species were formed this way, the end would not be precisely at the Flp cleavage site in FRT, but would be nearby.
Another model that would explain formation of tandem copies of the 2 μm DNA sequence is the double rolling circle model (Futcher, 1986
). However, this model does not account for the presence of apparent DSBs near FRT or for the participation of the HRR pathway in 2 μm hyperamplification in SUMO mutants. Both the single and double rolling circle models predict formation of primarily head-to-tail linkages between the tandem copies of 2 μm, if the circular template is a 2 μm monomer. This contrasts with our observation of equal levels of head-to-head and head-to-tail linkages in the HMW species. This discrepancy could be explained either if the template circle is a dimer or larger or if Flp activity randomizes the linkages after formation of the tandem multimer.
We did not detect evidence of ongoing replication of 2 μm in G2/M-arrested cells, suggesting that high levels of active RFs are not present in 2 μm throughout the cell cycle. The rolling circle intermediate proposed here could meet several fates. One is that Flp-dependent recombination between the circle and the tail could redirect the RF, and any HJ, off the end of the tail, generating a linear molecule. There could also be a cellular mechanism that destroys residual RFs late in the cell cycle. The absence of high levels of ongoing replication is consistent with a model where initiation of 2 μm hyperamplification is relatively rare, but that the aberrant species formed is replicated every cell cycle during S phase, so that the species persists in lineages where it develops. This model is consistent with the “nibbled” colony growth of cir+
SUMO pathway mutants, where some cir+
lineages grow well for many generations, indicating that these cells only rarely generate the species that causes growth arrest (Dobson et al., 2005
). The species predicted by our model would trigger checkpoint responses even if they are replicated only during S phase, because they would contain free ends without telomeres.
The SUMO pathway has complex effects on genome stability in S. cerevisiae
and directly affects diverse aspects of DNA metabolism, including DNA repair (Geiss-Friedlander and Melchior, 2007
). However, our work suggests that for 2 μm, the major effect of the SUMO pathway is to prevent Flp-dependent DNA damage, not to regulate the repair mechanism, as has been proposed (Burgess et al., 2007
). There is a slight discrepancy between our results, showing that mutants such as rad59
Δ have a modest effect on accumulation of the HMW species in siz1
Δ (), and a previous result showing that these mutants strongly suppress the 2 μm–related colony growth defects of the slx8
Δ mutant (Burgess et al., 2007
). It is likely that this discrepancy is explained by complete loss of 2 μm in the slx8
Δ experiment. In our hands, slx5
Δ and slx8
Δ strains rapidly self-select for loss of 2 μm (not shown). rad59
Δ also increased 2 μm loss in siz1
Δ (not shown). These observations emphasize the importance of using cir° versions of these SUMO pathway mutants for most experiments. Not only do cir+
versions of these mutants show apparent genome instability phenotypes that are actually secondary effects of 2 μm accumulation (), but they also display confusing effects resulting from spontaneous loss of the plasmid. Crossing the resulting cir° mutants to cir+
strains accentuates this confusion because it generates mutant segregants that have, at least temporarily, reacquired 2 μm and therefore grow more poorly than the parental cir° strain.