DNA is susceptible to a variety of insults from exogenous and endogenous sources. In contrast to DNA repair systems, which usually rely on the excision and subsequent re-synthesis of the damaged region to restore the original sequence information, DNA damage tolerance mechanisms allow the bypass of lesions without their actual removal. They ensure the progression of DNA replication on damaged DNA and are therefore essential for the survival of a cell in the presence of genotoxic agents. As lesion bypass is often associated with damage induced mutations, however, its activity needs to be tightly controlled. Our research aims at understanding the mechanisms and signals by which the ubiquitin and SUMO systems of posttranslational protein modification promote damage tolerance and limit the accumulation of unwanted mutations.
SUMO modification of budding yeast PCNA in S phase
SUMO is a posttranslational protein modifier of the family of ubiquitin-like proteins, common to all eukaryotes. Modification by SUMO has been implicated in the control of numerous biological processes, ranging from nucleo-cytoplasmic transport to the regulation of transcription, chromosome segregation and genome stability.
Our previous work has elucidated the mechanism of SUMO function in the case of budding yeast PCNA, the eukaryotic sliding clamp protein that ensures processive action of DNA polymerases during replication. We showed that PCNA sumoylation recruits an anti-recombinogenic helicase, Srs2, to replication forks by means of a preferential interaction of the helicase with the modified form of PCNA. By counteracting the formation of recombinogenic Rad51 filaments, Srs2 thus prevents unscheduled recombination events. When replication forks are stalled by DNA damage, the PCNASUMO-Srs2 system ensures a processing of the lesions via the damage bypass pathway that is triggered by the ubiquitination of PCNA.
Over the past year, our lab has investigated the architecture of the SUMO conjugates on PCNA as well as the regulation of PCNA sumoylation throughout the cell cycle. Purification of the SUMO conjugation machinery has allowed us to reconstitute the reaction in vitro and to complement our in vivo analysis with mechanistic insight into the modification reaction.
Architecture and assembly of poly-SUMO chains on budding yeast PCNA
Like other members of the ubiquitin family, SUMO is covalently attached to its target proteins by an enzymatic machinery comprising an activating enzyme (E1), a conjugating enzyme (E2) and a ligase (E3). The single E2, Ubc9, exhibits a marked preference for a short consensus motif, ΨKXE (where Ψ denotes a hydrophobic and X any amino acid). The predominant site of PCNA modification, K164, does not obey the consensus motif, despite being highly conserved among eukaryotic PCNA sequences. K127, which conforms to the consensus, is a minor site of attachment that is predominantly used when K164 is mutated. The ligase responsible for modification is Siz1, and SUMO is removed by the isopeptidase Ulp1.
We have analyzed the SUMO modification pattern of budding yeast PCNA and found that most aspects of our in vitro sumoylation reactions reflect the situation under physiological conditions. Two oligomeric SUMO chains of two to three moieties each, linked via internal sumoylation consensus motifs within the SUMO sequence, are assembled on PCNA. Siz1 both stimulates the overall efficiency of sumoylation and selects the modification site on PCNA. Furthermore, ubiquitin and SUMO chains are assembled independently, and we found evidence that both modifiers can coexist in vivo on a common PCNA subunit. Our results demonstrate for the first time the in vivo assembly of polymeric SUMO chains of defined linkage on a physiological substrate in yeast, but they also indicate that SUMO-SUMO polymers are dispensable for PCNASUMO function in replication and recombination.
Activation of S phase-dependent PCNA sumoylation by DNA
Modification by SUMO is often regulated by cellular signals that restrict the modification to appropriate situations. Nevertheless, most SUMO-specific ligases do not exhibit much target specificity, and ¿ compared with the diversity of sumoylation substrates ¿ their number is limited. This raises the question of how SUMO conjugation is controlled in vivo. We have now discovered an unexpected mechanism by which sumoylation of budding yeast PCNA is effectively coupled to S phase. We found that loading of PCNA onto DNA is both necessary and sufficient for sumoylation in vivo and greatly stimulates modification in vitro. Given that Siz1 contains a SAP domain, which in many other proteins mediates DNA binding, we argued that DNA would likely act as a template for bringing the E3 and the substrate into close proximity, thus enhancing the efficiency of the reaction. In fact, we were able to show that Siz1 binds to double stranded DNA by means of its SAP domain. To our surprise, however, DNA binding by Siz1 was not strictly required for efficient PCNA sumoylation. Instead, we found that the stimulatory effect of DNA on conjugation is mainly attributable to the loading of PCNA itself. These findings imply a change in the properties of PCNA upon DNA binding that enhances its capacity to be sumoylated, and we have indeed identified mutations in the inner, DNA contacting surface of PCNA that severely impair its capacity to be sumoylated. Hence, the loading of PCNA onto DNA during S phase provides a simple, yet effective mechanism to limit sumoylation to the relevant cell cycle stage (Figure 1).
Figure 1. Model for the role of DNA in the regulation of PCNA sumoylation. Transfer to DNA by the clamp loader RFC at the beginning of S phase induces a change in the properties of PCNA that facilitates its recognition by the SUMO conjugation machinery. Although Siz1 is normally associated with chromatin during G1 and S phase, DNA binding by the E3 is not strictly required for efficient modification of PCNA. At the end of S phase, unloading of PCNA may provide enhanced access to the isopeptidase Ulp1, situated at the nuclear pores. In addition, Siz1 leaves the nucleus at G2/M, thus further shifting the balance towards Ulp1-dependent deconjugation.
For a list of refereed research papers, see Publications (in navigation on left).
