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

Abstract

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.

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

“…Monoubiquitinated PCNA can be polyubiquitinated by the Rad5-Ubc13-Mms2 complex (Hofmann and Pickart, 1999) to allow error-free mechanisms (Torres-Ramos et al, 2002). Alternatively, K164 of PCNA can be SUMOylated to regulate recombination at the fork (Pfander et al, 2005;Moldovan et al, 2006) via a direct interaction with Srs2 (Pfander et al, 2005), a 3 -5 DNA helicase (Rong and Klein, 1993), thought to exert a negative control on HR (Krejci et al, 2003;Veaute et al, 2003).…”

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).…”