A decade has passed since SUMO (small ubiquitin-related modifier) was discovered to be a reversible post-translational protein modifier. During this time many enzymes that participate in regulated SUMO-conjugation and -deconjugation pathways have been identified and characterized. In parallel, the search for SUMO substrates has produced a long list of targets, which appear to be involved in most cellular functions. Sumoylation is a highly dynamic process and its outcomes are extremely diverse, ranging from changes in localization to altered activity and, in some cases, stability of the modified protein. At first glance, these effects have nothing in common; however, it seems that they all result from changes in the molecular interactions of the sumoylated proteins.

Concepts in sumoylation: a decade on

https://www.cell.com/cell/pdf/S0092-8674(07)00594-6.pdf

PCNA, the Maestro of the Replication Fork

The repair of DNA lesions that occur endogenously or in response to diverse genotoxic stresses is indispensable for genome integrity. DNA lesions activate checkpoint pathways that regulate specific DNA-repair mechanisms in the different phases of the cell cycle. Checkpoint-arrested cells resume cell-cycle progression once damage has been repaired, whereas cells with unrepairable DNA lesions undergo permanent cell-cycle arrest or apoptosis. Recent studies have provided insights into the mechanisms that contribute to DNA repair in specific cell-cycle phases and have highlighted the mechanisms that ensure cell-cycle progression or arrest in normal and cancerous cells.

Regulation of DNA repair throughout the cell cycle

Genome integrity is constantly monitored by sophisticated cellular networks, collectively termed the DNA damage response (DDR). A common feature of DDR proteins is their mobilization in response to genotoxic stress. Here, we outline how the development of various complementary methodologies has provided valuable insights into the spatiotemporal dynamics of DDR protein assembly/disassembly at sites of DNA strand breaks in eukaryotic cells. Considerable advances have also been made in understanding the underlying molecular mechanisms for these events, with post-translational modifications of DDR factors being shown to play prominent roles in controlling the formation of foci in response to DNA-damaging agents. We review these regulatory mechanisms and discuss their biological significance to the DDR.

/pdf/dynamics-of-dna-damage-response-proteins-at-dna-breaks-a-41jyz7dckm.pdf

Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications

Proteins of the small ubiquitin-related modifier (SUMO) family are conjugated to proteins to regulate such cellular processes as nuclear transport, transcription, chromosome segregation and DNA repair. Recently, numerous insights into regulatory mechanisms of the SUMO modification pathway have emerged. Although SUMO-conjugating enzymes can discriminate between SUMO targets, many substrates possess characteristics that facilitate their modification. Other post-translational modifications also regulate SUMO conjugation, suggesting that SUMO signalling is integrated with other signal transduction pathways. A better understanding of SUMO regulatory mechanisms will lead to improved approaches for analysing the function of SUMO and substrate conjugation in distinct cellular pathways.

The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition.

Damaged DNA, if not repaired before replication, can lead to replication fork stalling and genomic instability; however, cells can switch to different damage bypass modes that permit replication across lesions. Two main bypasses are controlled by ubiquitin modification of proliferating cell nuclear antigen (PCNA), a homotrimeric DNA-encircling protein that functions as a polymerase processivity factor and regulator of replication-linked functions. Upon DNA damage, PCNA is modified at the conserved lysine residue 164 by either mono-ubiquitin or a lysine-63-linked multi-ubiquitin chain, which induce error-prone or error-free replication bypasses of the lesions. In S phase, even in the absence of exogenous DNA damage, yeast PCNA can be alternatively modified by the small ubiquitin-related modifier protein SUMO; however the consequences of this remain controversial. Here we show by genetic analysis that SUMO-modified PCNA functionally cooperates with Srs2, a helicase that blocks recombinational repair by disrupting Rad51 nucleoprotein filaments. Moreover, Srs2 displays a preference for interacting directly with the SUMO-modified form of PCNA, owing to a specific binding site in its carboxy-terminal tail. Our finding suggests a model in which SUMO-modified PCNA recruits Srs2 in S phase in order to prevent unwanted recombination events of replicating chromosomes.

SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase

Homologous recombination (HR) is crucial for maintaining genome integrity by repairing DNA double-strand breaks (DSBs) and rescuing collapsed replication forks. In contrast, uncontrolled HR can lead to chromosome translocations, loss of heterozygosity, and deletion of repetitive sequences. Controlled HR is particularly important for the preservation of repetitive sequences of the ribosomal gene (rDNA) cluster. Here we show that recombinational repair of a DSB in rDNA in Saccharomyces cerevisiae involves the transient relocalization of the lesion to associate with the recombination machinery at an extranucleolar site. The nucleolar exclusion of Rad52 recombination foci entails Mre11 and Smc5-Smc6 complexes and depends on Rad52 SUMO (small ubiquitin-related modifier) modification. Remarkably, mutations that abrogate these activities result in the formation of Rad52 foci within the nucleolus and cause rDNA hyperrecombination and the excision of extrachromosomal rDNA circles. Our study also suggests a key role of sumoylation for nucleolar dynamics, perhaps in the compartmentalization of nuclear activities.

The Smc5–Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus

Homologous recombination is essential for genetic exchange, meiosis and error-free repair of double-strand breaks1. Central to this process is Rad52, a conserved homo-oligomeric ring-shaped protein, which mediates the exchange of the early recombination factor RPA by Rad51 and promotes strand annealing2,3. Here, we report that Rad52 of Saccharomyces cerevisiae is modified by the ubiquitin-like protein SUMO, primarily at two sites that flank the conserved Rad52 domain. Sumoylation is induced on DNA damage and triggered by Mre11–Rad50–Xrs2 (MRX) complex-governed double-strand breaks (DSBs). Although sumoylation-defective Rad52 is largely recombination proficient, mutant analysis revealed that the SUMO modification sustains Rad52 activity and concomitantly shelters the protein from accelerated proteasomal degradation. Furthermore, our data indicate that sumoylation becomes particularly relevant for those Rad52 molecules that are engaged in recombination.

Control of Rad52 recombination activity by double-strand break-induced SUMO modification

Modification of proteins by covalent attachment of ubiquitin and the ubiquitin‐like modifier SUMO are widespread regulatory events of all eukaryotic cells. SUMOylation has received much attention, because several identified targets play prominent roles, in particular, in cell signaling, gene expression, and DNA repair. Notably, only a very small fraction of a substrate is usually SUMOylated at steady‐state levels, which could be because modification is reversible and transient. Because of the low level of modification, SUMOylated proteins are often overlooked or sometimes misinterpreted as a less important fraction of a protein pool. Here we discuss procedures that can circumvent identification problems and describe methods for their verification.

Meik Sacher

Papers

SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase

The Smc5–Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus

Control of Rad52 recombination activity by double-strand break-induced SUMO modification

Identification of SUMO-protein conjugates.