Crosstalk between SUMO and Ubiquitin on PCNA Is Mediated by Recruitment of the Helicase Srs2p

Abstract

Posttranslational modification of proliferating cell nuclear antigen (PCNA), an essential processivity clamp for DNA polymerases, by ubiquitin and SUMO contributes to the coordination of DNA replication, damage tolerance, and mutagenesis. Whereas ubiquitination in response to DNA damage promotes the bypass of replication-blocking lesions, sumoylation during S phase is damage independent. As both modifiers target the same site on PCNA, an antagonistic action of SUMO on ubiquitin-dependent DNA damage tolerance has been proposed. We now present evidence that the apparent negative effect of SUMO on lesion bypass is not due to competition with ubiquitination but is rather mediated by the helicase Srs2p, which affects genome stability by suppressing unscheduled homologous recombination. We show that Srs2p physically interacts with sumoylated PCNA, which contributes to the recruitment of the helicase to replication forks. Our findings suggest a mechanism by which SUMO and ubiquitin cooperatively control the choice of pathway for the processing of DNA lesions during replication.

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

“…At the C‐terminus, Srs2 contains a variety of regulatory motifs, which are modulated through post‐translational modifications and/or required for the interactions of Srs2 with other proteins, including PCNA, and these are important for its role at replication forks (Papouli et al , 2005; Pfander et al , 2005; Burgess et al , 2009) and regulation of the D‐loop extension (Burkovics et al , 2013). However, most of the C‐terminus was not required for the role of Srs2 in DSB repair via de novo telomere addition, BIR and SSA (Fig 8A–D).…”

“…In contrast, the Srs2 ATPase activity was important for all the repair mechanisms analysed (Fig 8B–D). Therefore, the role of Srs2 in DSB repair is different from its role at replication forks and does not require Srs2–PCNA interaction (Papouli et al , 2005; Pfander et al , 2005). Instead, Srs2 acts upstream of PCNA by removing Rad51 from DNA repair loci in order to stimulate PCNA recruitment, thereby promoting the speed with which ssDNA is converted into its functional double‐stranded form.…”

“…The Srs2 helicase inhibits HR machinery by disassembling Rad51 filament and reducing DNA extension, as demonstrated in vitro (Burkovics et al , 2013; Krejci et al , 2003; Veaute et al , 2003). This function is believed to be important for repression of excessive recombination, particularly at replication forks where Srs2 is recruited and regulated through its C‐terminal domain (Papouli et al , 2005; Pfander et al , 2005; Burgess et al , 2009). Loss of Srs2 results in a paradoxical phenotype.…”

Genetic evidence for a role of Saccharomyces cerevisiae Mph1 in recombinational DNA repair under replicative stress

“…mph1 and siz1 mutations confer additive DNA damage sensitivity towards camptothecin, MMS and 4-NQO as determined by drop dilution assays Srs2, recruited to the replication fork by means of a direct interaction with SUMO modified PCNA, acts as a guarding mechanism to regulate recombination during replication (Papouli et al, 2005;Pfander et al, 2005). If this recruitment is relevant in the functional interaction with Mph1, deletion of the SIZ1 gene, coding for the ligase responsible for PCNA SUMOylation (Pfander et al, 2005), should have a synthetic interaction with mph1, similar to that of srs2.…”

DNA Replication to Aid Somatic Hypermutation

“…This mutated PCNA cannot be ubiquitinated (Kannouche et al, 2004) and thereby would be unable to activate TLS polymerases (Bienko et al, 2005). PCNA also undergoes polyubiquitination and sumoylation in yeast (Papouli et al, 2005;Pfander et al, 2005), suggesting the existence of more posttranslational modifications of the mammalian PCNA that can play a role in the SHM polymerase switch. Negative regulators of the polymerase switch would include the deubiquitinase USP1, whose degradation correlates with the accumulation monoubiquitinated PCNA in mammalian cells (Huang et al, 2006).…”