Elg1, an alternative subunit of the RFC clamp loader, preferentially interacts with SUMOylated PCNA

Abstract

Replication-factor C (RFC) is a protein complex that loads the processivity clamp PCNA onto DNA. Elg1 is a conserved protein with homology to the largest subunit of RFC, but its function remained enigmatic. Here, we show that yeast Elg1 interacts physically and genetically with PCNA, in a manner that depends on PCNA modification, and exhibits preferential affinity for SUMOylated PCNA. This interaction is mediated by three small ubiquitin-like modifier (SUMO)-interacting motifs and a PCNA-interacting protein box close to the N-terminus of Elg1. These motifs are important for the ability of Elg1 to maintain genomic stability. SUMOylated PCNA is known to recruit the helicase Srs2, and in the absence of Elg1, Srs2 and SUMOylated PCNA accumulate on chromatin. Strains carrying mutations in both ELG1 and SRS2 exhibit a synthetic fitness defect that depends on PCNA modification. Our results underscore the importance of Elg1, Srs2 and SUMOylated PCNA in the maintenance of genomic stability.

Figure 1

Genetic interactions between ELG1 and PCNA depend on PCNA modification. Serial dilutions of yeast cultures on minimal SD-complete plates with or without methylmethane sulphonate (MMS). Ten different MMS concentrations were used for each experiment; the informative ones are shown. (A) Deletion of ELG1 suppresses the sensitivity of Δrad5 to DNA damage. (B) elg1 suppression effect cannot be seen on a pol30-RR background. (C) elg1 suppression effect cannot be seen in the absence of the Siz1 SUMO ligase.

Figure 2

Elg1 interacts physically with PCNA and SUMO. (A) Two-hybrid interaction between Elg1 fused to GAL4-binding domain (BD) and a number of constructs containing PCNA or SUMO fused to the GAL4-activating domain (AD). (B) Co-IP of an Elg1-13Myc protein with PCNA. After IP with IgG as a negative control or with anti-PCNA antibody, western blots were carried out with anti-PCNA or anti-myc antibodies. (C) Co-IP of an Elg1-13Myc protein with Histidine-tagged PCNA in ulp1-1 strains. The yeast culture was treated with 0.3% MMS for 1 h before lysis. The asterisk denotes a faint additional SUMOylation of PCNA at an unknown location that is sometimes detected.

Figure 3

The N-terminus of Elg1 interacts with PCNA and SUMO. (A) Two-hybrid experiment with SUMO fused to the GAL4-activating domain (AD) and segments of ELG1 fused to the GAL4 DNA-binding domain (BD). (B) The same experiment as in (A) except that Pol30-RR is fused to AD. (C) Pull-down experiment with recombinant N-terminus of Elg1 fused to GST or with GST alone. The recombinant protein was mixed with lysates of yeast containing HIS-PCNA. Cells were treated with different amount of MMS as indicated. (D) Pull-down experiment with recombinant N-terminus of Elg1 fused to GST and yeast lysates (from cells treated with 0.3% MMS for 1 h) in which HIS-PCNA is mutated at the lysine that can undergo modification. (E) Quantitation of the relative amount of modified PCNA in the WCE and in the pellet (based on three experiments). (F) In vitro SUMOylation of PCNA was performed with purified Aos1p-Uba2p (E1), Ubc9 (E2), Siz1 (E3), PCNA, SUMO and ATP. The left panel shows the full reaction, as well as control reactions each missing one component. The right panel shows a pull-down experiment with recombinant N-terminus of Elg1 fused to GST or with GST and purified SUMOylated PCNA obtained in vitro.

Figure 4

Higher levels of unmodified or SUMOylated PCNA in the chromatin of elg1 mutant cells. Wild-type or elg1 cells containing HIS-tagged PCNA were subjected to chromatin fractionation and Ni-NTA pull down after treatment without or with MMS for 60 min., followed by immunoblotting. (A) Unmodified or SUMOylated PCNA was visualized by western blotting using antibodies against SUMO or PCNA. PGK was used as a non-chromatin marker, whereas Acetylated Histone H4 (AcH4) was used as a chromatin marker. Whole cell extract (WCE), supernatant (Sup) and chromatin (CHR) fractions are shown. (B) Quantitation analysis of the amount of SUMOylated or unmodified PCNA of wt and elg1 mutants without MMS. The amount of PCNA or modified PCNA in the chromatin fraction was divided by the amount of AcH4 signal in the chromatin fraction. The average of four experiments is presented. (C) Quantitation analysis (as in B) of the samples treated with 0.3% MMS.

Figure 5

Synthetic interactions of elg1 with Srs2 (A) Higher levels of Srs2 in the chromatin fraction of Δelg1 cells. Wild-type or elg1 cells containing Srs2-Myc were subjected to chromatin fractionation after treatment without or with 0.02% MMS for 60 min. PGK was used as a non-chromatin marker, whereas AcH4 was used as chromatin marker. (B) elg1 and srs2 show a synthetic fitness phenotype. Tetrad analysis of a diploid heterozygote srs2/+, +/elg1. (C) pol30-RR partially suppresses the synthetic sickness of elg1 srs2 mutants. Tetrad analysis of a diploid homozygote for elg1 (elg1/elg1) and heterozygote srs2/+, pol30-RR/+. (D) pol30-RR partially suppresses the sensitivity of elg1 srs2 to MMS. (E) rad18 (no ubiquitination of PCNA) does not suppresses the sensitivity of elg1 srs2 to MMS.

Figure 6

SIM and PIP motifs mediate the interaction between Elg1, PCNA and SUMO. Interaction between different fragments of the N-terminus of ELG1 fused to GAL4 BD and (A) SUMO or (B) PCNA-RR fused to GAL4 AD. (C) The location of the PIP box (bright grey) and SIM motifs (dark grey) in the N-terminus of Elg1. (D) Sequence alignment between proteins that contain PIP boxes. (E) Elg1 interactions with SUMO and PCNA are reduced in SIM and PIP mutants, accordingly. Two-hybrid interactions between SUMO or PCNA-RR and the N-ter Elg1 carrying various mutations. The SIM alleles were I28A (SIM1), I93K (SIM2), II121, 122AA (SIM3) and combinations thereof; the PIP mutants were either SV57, 58AA (PIP57) or VV58, 59 (PIP58). (F) Pull down with the N-terminus of elg1 with or without mutation in the three SIM motifs. The yeast lysates were treated with 0.3% MMS for 1 h.

Figure 7

The SIMs and PIP motifs in ELG1 are important for its activity. (A) The interaction of Elg1 with SUMOylated PCNA is mediated by the SIM and PIP motifs. A Co-IP experiment with ELG1 mutants or wt cells tagged with 13-MYC or in strains in which ELG1 is not tagged (as a control). The SIM allele used is the triple mutant I28A, I93K, II121, 122AA; the PIP allele is SV57, 58AA, and the SIM-PIP (SP) allele is a combination of all the mutations. The yeast lysates were treated with 0.3% MMS for 1 h. (B) Western blot analysis to detect the protein amount of Elg1 mutants in vivo using anti-Myc antibody or anti-PGK antibody as loading control. (C, D) Sensitivity to MMS of elg1 mutants in RAD5 and rad5 mutant background, accordingly. (E) No effect on the sensitivity to MMS by elg1 mutants could be observed in a pol30-RR background.