SUMO as a “reset button” for PCNA functions

PCNA is a fundamental regulator of DNA replication and replication-linked activities, acting as a docking site for proteins required at the replisome (Maga and Hubscher, 2003; Tsurimoto, 1999; Warbrick, 2000). Most of the proteins that bind PCNA contain a common motif, the PIP-box, which represents a PCNA-docking peptide. Proteins shown so far to use their PIP-box for PCNA

interaction include DNA polymerases , and , FEN-1, DNA ligase I, RFC,

CAF-1, WSTF, Dnmt1, MSH3, MSH6, MLH1, EXO1, APE2, UNG2, MPG, XPG, Gadd45, p21, Topoisomerase II, ING1, WRN, Rrm3, UNG2, Cdt1, Mcm10 (see paragraph 1.2.2) and Eco1 (this study). All these proteins bind to a hydrophobic groove of PCNA, formed by the IDCL and the C-terminal part (Gulbis et al., 1996; Matsumiya et al., 2002), and they are likely to compete with each other for PCNA binding. Indeed, p21 binding to PCNA inhibits cell

3.3 Control of sister chromatid cohesion by PCNA and SUMO

proliferation by blocking the access of DNA polymerases (Gulbis et al., 1996; Wagaet al., 1994).

Even though a PCNA trimer has three docking sites for PIP peptides, the sheer number of interactors suggests that cells have to regulate the competition of PIP-box proteins. One way to achieve this is by altering the sequences of the PIP-boxes. Indeed, the affinity of a PIP-box binding to the hydrophobic cleft of PCNA can be dramatically altered by even modest changes in the identity of the non-conserved aminoacids in the QxxL/I/MxxFF/Y box, as well as of those N- or C-terminal of the this sequence (Bruning and Shamoo, 2004). These sequence alterations result in a range of PCNA binding affinities calculated to vary at least 200-fold. The p21 PIP-box apparently has the highest affinity for PCNA, explaining its potent ability to

effectively block replication. Therefore, the in vivo dynamics of PCNA

interactions might be regulated by fine-tuning the affinities of PIP-boxes. (Bruning and Shamoo, 2004). Another model implies the kinase Cdk2 in regulating PCNA interactions (Prosperi, 2006). Interestingly, Cdk2 is one of the few PCNA interactors lacking a PIP-box, and was found in trimeric

complexes comprising PCNA and PIP-box proteins (Riva et al., 2004). Cdk2

phosphorylation of PIP-box proteins like RFC-1, FEN1 and DNA ligase I was shown to trigger their dissociation from the complex (Henneke et al., 2003), and presumably this allows a new interactor to bind PCNA. Interestingly, a large-scale proteomic study (Ubersaxet al., 2003) identified Eco1 as a Cdc28

(the S. cerevisiae homolog of Cdk2) target, suggesting that the Cdk-

dependent regulatory mechanism might apply as well to the PCNA-Eco1 interaction.

Genetic and biochemical analyses presented in the study at hand evidence that inS. cerevisiae, PCNA modification by SUMO inhibits cohesion by blocking the binding of Eco1 to PCNA (see paragraph 3.2.4). Intriguingly, PCNA SUMOylation appears to inhibit PCNA-linked functions more broadly. Genetic assays revealed that PCNA SUMOylation also interferes with the functions of two other PIP-box-containing PCNA interactors, Rfc1 and Cac1 (Figure 28). Remarkably, K127, one of the SUMO acceptor lysine residues for PCNA SUMOylation resides within the IDCL, an area of PCNA used for PIP- box binding. Moreover, K127 is encompassed in a “consensus” site for

SUMOylation, defined by the sequence KxD/E, which is known to directly

bind Ubc9, the E2 SUMO-conjugating enzyme. Indeed, Ubc9 binds PCNA with high affinity and can efficiently displace Eco1 from PCNA (Figure 27). Therefore, it is conceivable that Ubc9 binding and PCNA SUMOylation might in general interfere with the function of proteins that bind PCNA via this region. Thus, SUMO modification of PCNA might constitute yet a third

Discussion

mechanism for coordinating PIP-box binding to PCNA. SUMO may function as a “reset button” that clears PCNA from its binding partner in order to facilitate another round of engagement, with a new co-factor.

Lysine 164 of PCNA is located further away from the PIP-box binding site, and it is therefore not expected to markedly affect the binding of PIP-box proteins. Indeed, although K164 is the acceptor site for most of PCNA

SUMOylation (Hoege et al., 2002), mutation of this lysine had only minor

effects on cohesion (Figure 25A,B) and Rfc1-dependent replication (Figure 28A), and no detectable effect on Cac1-dependent cohesion (Figure 28B). In contrast, the low levels of SUMOylation at K127, located in the IDCL, strongly suppress the functions of Eco1, Rfc1 and Cac1 in genetic experiments (Figures 25, 28). Previously, PCNA SUMOylation was shown to inhibit recombination by recruiting the helicase Srs2 (Papouliet al., 2005; Pfander et al., 2005). Srs2 binding to PCNA and recombination inhibition is proportional to the amount of SUMOylated PCNA, thus K164 is much more relevant than K127 for this function (Pfander et al., 2005). Altogether, these observations show that SUMOylation at K127 and K164 of PCNA are not totally equivalent. Because only K164 SUMOylation depends on the SUMO E3 ligase Siz1 (Pfander et al., 2005), this enzyme may delicately balance these two SUMO- dependent PCNA functions. Understanding the mechanism of Siz1 regulation should unravel how SUMO coordinates the numerous events that are linked to replication.

Intriguingly, K127 is not conserved in metazoans. Besides S .

cerevisiae, SUMOylation of PCNA was reported so far in Xenopus egg extract (Leach and Michael, 2005) and in chicken DT40 cells (Figure 8), and in both cases it affected K164. Therefore it is possible that the K127-SUMO mechanism for regulating the dynamics of PIP-box interactions is not conserved. On the other hand, as in S. cerevsiae the levels of SUMOylation at K127 are very low, it cannot be excluded that this regulation is present in other eukaryotic cells. Perhaps a lysine from the IDCL or its vicinity is modified by SUMO in metazoans, to levels undetectable with the technologies currently employed.

In conclusion, this study identified inhibition of sister chromatid cohesion establishment as a novel function of PCNA SUMOylation. Most likely, SUMO performs this activity by blocking the access of the cohesion factor Eco1 to PCNA, thereby inhibiting an interaction essential for cohesion establishment. Repression of cohesion can be added to the list of three previously known functions of PCNA modifications (Figure 30): two types of DNA repair, (1) error-prone and (2) error-free, mediated by ubiquitin modification and (3) recombination inhibition, mediated by SUMOylation.

3.3 Control of sister chromatid cohesion by PCNA and SUMO

Especially relevant for cohesion inhibition is SUMO modification at K127 of PCNA. Moreover, K127 SUMOylation may represent a more general mechanism to regulate PCNA functions, by removing a bound cofactor and thus resetting PCNA for a new interaction.

PCNA modifications with ubiquitin and SUMO show how handy these posttranslational modifications can be for regulating protein functions, as they can act both by enabling and by inhibiting protein-protein interactions. By employing local control on the conjugation/deconjugation machineries, cells can turn on and off the pathway controlled by modification in a fast and energetically cheap way. Thus, it is of no wonder that more and more proteins are found to be posttranslationally modified by ubiquitin and/or UBLs, and it is becoming clear that cells use such modifications for regulation of many biological functions. PCNA serves as a perfect example for the versatility of the ubiquitin/UBL system and constitutes an excellent model for studying cellular regulation by posttranslational protein modifications.

Figure 30. The ubiquitin/SUMO switch. PCNA functions are modulated by

posttranslational modifications. While two types of ubiquitylation activate DNA repair pathways, modifications by SUMO inhibit recombination, cohesion and possibly other PCNA functions.

Materials and methods