Recent studies have implicated sumoylation in coping with DNA damage. To provide a systematic view of the contribution of sumoylation to the DNA damage response, we show that, in parallel to checkpoint phosphorylation, cells also induce sumoylation of many proteins needed for replication and repair in the presence of DNA damage. Consistent with a broader role for sumoylation, we found that effective sumoylation is important for replication under damage conditions and DSB end processing, as well as survival in the presence of genotoxins. Moreover, sumoylation induction does not require the key checkpoint kinases, and sumoylation and the Mec1 checkpoint function largely autonomously in MMS conditions. These findings support a model in which DNA damage-induced sumoylation (or DDIS) is an integral part of the DNA damage response, and the effects of checkpoint phosphorylation and sumoylation combine to generate a robust response to DNA damage ().
Our identification of many SUMO substrates at endogenous expression levels was made possible by biochemical examination of individual proteins. Because the sumoylation/desumoylation cycle is highly dynamic, and because sumoylated forms are prone to deconjugation during purification and are refractory to mass spectrometry-based proteomic approaches, identifying SUMO substrates has thus far been challenging (Wilson and Heaton, 2008
; Jeram et al., 2009
). Immunoprecipitating individual tagged proteins provides an alternative, perhaps a more sensitive and direct way to detect endogenous protein sumoylation, although we do not exclude the possibility that some substrates still elude detection. Quantification of a quarter of the substrates that exhibited moderate to high levels of sumoylation showed that the percentage of sumoylated forms ranged from 2% to almost 20% of the protein (see Experimental Procedures). In addition, specific sumoylation patterns were associated with each substrate, varying from monosumoylation (e.g., Mcm4, Mcm5, and Mlh1 in ) to poly- or multisumoylation (e.g., Mcm2, Mcm6, and Rad25 in ). While it is not entirely clear how these SUMO forms can differentially affect protein functions, poly- or multisumoylation may confer stronger affinity to proteins containing multiple SUMO interaction domains, thus favoring these interactions (Bruderer et al., 2011
). Regardless of the forms of SUMO modification, sumoylation of some of the new targets likely modulates replication or repair processes, in light of the defects shown by mutants with global reductions in sumoylation. This extensive list of SUMO substrates enables future work to elucidate the specific roles of sumoylation in individual genome protection programs.
Our results suggest that cells can sense the insults from DNA damaging agents and respond by increasing protein sumoylation. We have identified MRX as a positive regulator of a subset of DNA damage-induced sumoylation events, specifically affecting proteins involved in recombinational repair (, and S2F
). Because the end clipping function but not Tel1 activation is required for sumoylation induction (), MRX may exert a local effect at DSBs or DNA lesions undergoing recombination. Since the role of MRX in supporting sumoylation appears to be targeted to a small group of substrates, cells likely rely on additional proteins besides MRX to induce the modification of other functional groups of SUMO substrates.
Additional positive regulators of DDIS are unlikely to include the canonical checkpoint kinase Mec1 because removal of Mec1 had a negligible effect on the sumoylation of all 16 proteins tested following treatment with a high concentration of MMS (e.g., ). At lower concentrations of MMS, sumoylation of several repair proteins, particularly those involved in DSB repair, even increased in mec1
Δ cells (). This could be due to a higher level of DNA lesions, such as DSBs, present in mec1
Δ cells (Cha and Kleckner, 2002
) or could be related to Mec1's known suppression of recombination foci (Lisby et al., 2004
; Alabert et al., 2009
); it may also be a compensatory effect, with increased sumoylation buffering the repair defects of checkpoint mutants. This effect might become less noticeable at high doses of MMS, when sumoylation would already be strongly induced in wild-type cells. The separation between DDIS and the Mec1 checkpoint is demonstrated by the finding that simultaneous defects in both SUMO and checkpoint pathways exacerbate DNA damage sensitivity (). However, there is a certain amount of interplay within the framework of SUMO and checkpoint responses working alongside each other. For example, the minor checkpoint defects in sumoylation mutants upon MMS treatment may indicate that the sumoylation of some DNA lesion detector proteins could contribute to optimal checkpoint activation (, and ). In addition, a stronger impact on checkpoint activation by sumoylation was detected in the presence of DSBs, due to the effect on resection ( and S2B
In summary, we present evidence that DDIS occurs on a large scale, that effective sumoylation is important for coping with DNA damage, and that induced sumoylation requires at least one known DNA damage sensor but not key checkpoint kinases. Together, these data support the idea that a SUMO-based response is an integral part of the DDR and acts alongside checkpoint signaling to boost cells' ability to replicate and repair DNA under damage conditions. This work is likely to have important implications in human cells because sumoylation and pathways governing genomic stability are highly conserved between yeast and humans. Indeed, human homologs of some of the SUMO substrates identified here were recently found to be sumoylated (Golebiowski et al., 2009
; ). Future work examining the interplay between the SUMO and checkpoint responses in yeast and humans will lead to a more comprehensive picture of the DNA damage response. Given the increased complexity associated with both sumoylation and the checkpoint response in human cells, their relationship may be more intricate than in yeast. However, if these two systems make largely separable contributions, as seen here, an approach simultaneously targeting both these branches of the DDR, or ablating DDIS in checkpoint-deficient cells, may lead to more effective cancer treatment strategies.