Journal of Molecular Biology
Volume 376, Issue 1, 8 February 2008, Pages 221-231
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Architecture and Assembly of Poly-SUMO Chains on PCNA in Saccharomyces cerevisiae

https://doi.org/10.1016/j.jmb.2007.12.008Get rights and content

Abstract

Posttranslational modifications of proliferating cell nuclear antigen (PCNA), the eukaryotic processivity clamp for DNA polymerases, regulate the pathways by which replication problems are resolved. In the budding yeast Saccharomyces cerevisiae, ubiquitylation of PCNA in response to DNA damage facilitates the replicative bypass of lesions, whereas conjugation of the ubiquitin-related modifier (SUMO) prevents unscheduled crossover events during S phase. We have analyzed the SUMO modification pattern of budding yeast PCNA in vivo and in vitro and found that most aspects of our in vitro sumoylation reactions reflect the situation under physiological conditions. We show that two oligomeric SUMO chains of two to three moieties each, linked via internal sumoylation consensus motifs within the SUMO sequence, are assembled on PCNA. The SUMO-specific ligase 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.

Introduction

Posttranslational modifications reversibly alter protein function by modulating inter- or intramolecular interaction surfaces, thereby affecting the activity, stability or localization of the modified protein. Among the various modifiers, members of the ubiquitin family are particularly versatile with respect to the functional consequences of the modification for a given target.1 They are attached to their substrates by a series of enzymes: activating enzymes (E1) that activate the modifier in an ATP-dependent manner, conjugating enzymes (E2) that transfer the activated modifier from the E1 to the substrate, and ligases (E3) that convey selectivity to the conjugation reaction.1, 2

Ubiquitin itself is best known for its role as a degradation signal when attached in polymeric chains where the carboxy (C) terminus of one ubiquitin is linked to lysine (K) 48 of the preceeding moiety.3, 4 However, other chain geometries that appear to convey alternative signals in kinase activation or DNA damage bypass have also been identified, and monoubiquitylation is implicated in yet other biological functions, such as endocytosis, intracellular vesicle transport and the modulation of chromatin structure.5, 6, 7 Like ubiquitin, the small ubiquitin-related modifier (SUMO)8 can form polymeric chains. In higher eukaryotes, at least three different SUMO proteins are found in conjugates, and among these, SUMO-2 and -3 bear an internal motif, ΨKX(D/E) (where Ψ represents a bulky aliphatic residue and X any amino acid), that can be recognized by the SUMO-specific E2, Ubc9, as a modification site.9, 10, 11 Accordingly, in vitro sumoylation reactions readily give rise to poly-SUMO chains. Yeast SUMO, encoded by the SMT3 gene, bears three potential sumoylation sites, K11, K15 and K19. Detailed in vitro studies have shown that poly-SUMO chains can be formed via these lysines on the conjugating enzymes themselves, on model substrates or free in solution.12, 13, 14, 15 In contrast, in vivo chain formation is rather poorly characterized.16, 17 In addition to unanchored oligomeric chains, high molecular weight species have been observed, but it remains unclear whether these represent free polymeric chains or large protein conjugates.12 More importantly, the biological function of poly-SUMO chains remains unknown. On one hand, preventing SUMO polymerization causes defects in the assembly of the synaptonemal complex during meiosis in budding yeast.18 On the other hand, unphysiological accumulation of polymeric chains may also have adverse effects for the cell, as deletion of a SUMO-specific isopeptidase, Ulp2, which is responsible for disassembling large SUMO polymers in yeast, results in chromosome instability and sensitivity to DNA-damaging agents.12, 19

Finally, some proteins are subject to modification by both SUMO and ubiquitin.20 A prominent example is proliferating cell nuclear antigen (PCNA), the homotrimeric processivity clamp for replicative DNA polymerases. Mono- and polyubiquitylation at a highly conserved lysine, K164, activates the bypass of replication-stalling DNA lesions by translesion synthesis or an error-free damage-avoidance mechanism in many eukaryotic organisms.21, 22, 23, 24, 25, 26, 27, 28 In contrast, PCNA sumoylation is observed in a limited number of species.29 In S. cerevisiae the modification is mediated by the SUMO ligase Siz1 and causes the recruitment of the helicase Srs2 to replication forks during S phase in order to prevent unwanted crossover events.30, 31, 32

We have now undertaken an analysis of PCNA modification patterns in budding yeast. We show that the E3 Siz1 assembles two oligomeric SUMO chains on PCNA, linked via the internal sumoylation consensus motifs, and it appears that ubiquitin and SUMO can coexist on a single PCNA subunit. The effect of Siz1 is largely reproduced by an in vitro sumoylation system using recombinant components. Surprisingly, in this assay the presence of a second sumoylation site stimulates poly-SUMO chain formation. Our results demonstrate for the first time the in vivo assembly of polymeric chains on a physiological SUMO substrate in yeast, but they also indicate that the SUMO–SUMO linkages are dispensable for PCNASUMO function, implying that SUMO is fully active as a monomer on its target, PCNA.

Section snippets

PCNA is modified by poly-SUMO chains in vivo

Sumoylation of PCNA from S. cerevisiae occurs primarily on the conserved lysine residue K164, which does not conform to the sumoylation consensus motif, and to some extent on K127 within the sumoylation consensus sequence “LKIE.”21 Modification at K164 requires the SUMO ligase Siz1.21, 22 After isolation of His-tagged PCNA under denaturing conditions from extracts of exponentially growing cultures, the two modified forms could be distinguished on anti-PCNA Western blots based on their distinct

The architecture of PCNA modifications

Despite the propensity of Ubc9 to polymerize SUMO in vitro, few examples of poly-SUMO chains attached to physiological substrates have been described in vivo. In mammalian cells, a di-SUMO conjugate of HDAC4 was identified after cotransfection of plasmids encoding SUMO-2 and the substrate.17 In yeast, higher order polymers, although observable, have not been assigned to any particular substrate. We have now shown that budding yeast PCNA is modified in vivo by two short SUMO chains under

Plasmids and yeast strains

Yeast expression vectors for PCNA and HisPCNA are based on the integrative plasmids YIplac128 or YIplac211,43 into which the POL30 open reading frame was cloned as a SmaI/PstI fragment with or without an N-terminal His6 epitope (BamHI/SmaI) in combination with a PCR product covering 524 bp of the POL30 upstream sequence (EcoRI/BamHI). A transcriptional terminator derived from ADH1 (PstI/SphI) was used for the YIplac128-based vectors, and 1131 bp of the POL30 3′ sequence (PstI/PstI) were

Acknowledgements

Our special thanks go to Diana Huttner for early contributions to this project, experimental advice and helpful discussions. We thank Jo Parker for analysis of the K196R mutant, Andrea Bucceri and Adelina Davies for investigating PCNA–Siz1 and Katharina Heidrich for PCNA–Ubc9 interactions, Eva Böttcher, Angelika Jacobs and Sandra Pahnke for technical assistance and Erica Johnson for providing numerous strains and expression constructs. This work was supported by Cancer Research UK and an EMBO

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    Present address: H. Windecker, The Friedrich Miescher Laboratory of the Max Planck Society, Spemannstrasse 39, D-72076 Tübingen, Germany.

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