Ubiquitylation of PCNA is required for the UBZ-dependent activity of Mgs1. (A) UBZ-dependent sensitization to DNA-damaging agents by MGS1 overexpression requires PCNA ubiquitylation. MMS sensitivity assays were performed with the indicated strains harbouring Gal-MGS1 or Gal-MGS1*, as described in Figure 1E. The empty vector served as control. (B) The effect of MGS1 overexpression on DNA damage sensitivity is mediated by a combination of TLS and error-free damage bypass. MMS sensitivities upon MGS1 or MGS1* overexpression were assayed as in A in strains lacking the capacity for either error-free damage bypass mediated by UBC13-dependent PCNA polyubiquitylation (ubc13) or monoubiquitin-dependent TLS by damage-tolerant polymerases (rad30 rev1 rev3, designated as tlsΔ), or both pathways. (C) MGS1 deletion or overexpression does not affect PCNA ubiquitylation. Strains harbouring His₆-tagged POL30 alleles and either an mgs1 deletion or the indicated vectors were incubated with or without 0.02% MMS for 60 min, and PCNA and its modified forms were isolated by Ni–NTA affinity chromatography under denaturing conditions and detected by western blotting.

The UBZ domain contributes to the recruitment of Mgs1 to stalled replication intermediates. Association of Mgs1^9myc at an early replication origin was monitored by ChIP and quantitative PCR. G1-arrested cells were released into the cell cycle in the presence of 100 mM hydroxyurea to slow the progression of replication forks and induce PCNA ubiquitylation, and association of Mgs1^9myc with an early-firing replication origin (ARS607) was assessed at 10-min intervals (right panel). ChIP assays of PCNA from the same cultures served as a positive control for a fork-associated protein (left panel), and ChIP assays at a late origin (ARS501) that does not fire under these conditions served as a negative control. Signals were quantified relative to background samples prepared without antibody. Error bars indicate standard deviations from two to three experiments. (A) Mgs1 expressed at native levels is barely detectable by ChIP. Shown are signals of PCNA (left) and Mgs1^9myc (right) expressed at physiological levels. (B) When overexpressed, Mgs1 is detectable at stalled replication intermediates. Shown are ChIP signals of PCNA (left) and overexpressed Mgs1^9myc (right). (C) Mutating the UBZ domain of Mgs1 or the ubiquitylation site on PCNA reduces the association of Mgs1 with replication intermediates. Shown are the ChIP signals of PCNA and Mgs1^9myc in the indicated strains at ARS607. The signals at ARS501 are omitted for clarity.

Mgs1 competes with Pol32 for PCNA binding in the three-hybrid system. Fusions of the Gal4 activating domain to PCNA*, Ub*–PCNA* or Ub*₄–PCNA* (as indicated above the panels) were combined with fusions of the DNA-binding domain to Pol32 as described in Figure 3A, and the effect of overexpressing MGS1 or MGS1* was assessed by spot assays, using 10-fold dilutions of three independent cultures per strain. An empty vector served as control. Uracil was omitted from the plates to maintain the additional plasmid (-LWU, -HLWU).

The UBZ domain is required specifically for the damage-related activities of Mgs1. (A) The UBZ domain is dispensable for longevity. Life span analysis of WT cells in comparison with mgs1 deletion mutants alone or complemented with either MGS1 or the UBZ mutant (D31A) allele, MGS1*, on an integrative plasmid under control of its own promoter. (B) The UBZ domain does not influence spontaneous recombination. Rates of heteroallelic recombination in the HIS1 locus (his1-1/his1-7) in diploid mgs1/mgs1 cells complemented with two copies of MGS1 or MGS1* as indicated. The empty vector served as control. Error bars indicate standard deviations. (C) The UBZ domain is irrelevant for the synthetic lethality of rad18 mgs1 mutants. Shown are progeny of genetic crosses between rad18 and mgs1 mutants, each carrying an empty vector, MGS1 or MGS1* as indicated. Tetrads are aligned in perpendicular orientation. (D) The UBZ domain is irrelevant for the synthetic growth defect of sgs1 mgs1 mutants. Growth of the indicated strains on rich medium was monitored by spot assays. (E) Overexpression of MGS1 sensitizes cells to DNA damage and replication problems in a UBZ-dependent manner. Spot assays for sensitivity to MMS and hydroxyurea (HU) at the indicated concentrations were performed with WT cells carrying vectors for overexpression of MGS1 alleles under control of a galactose-inducible promoter (Gal-MGS1 and Gal-MGS1*). The empty vector served as control. (F) Overexpression of MGS1 suppresses DNA damage-induced mutagenesis in a UBZ-dependent manner. Frequencies of MMS-induced mutations in the CAN1 locus were determined for WT strains harbouring Gal-MGS1 or Gal-MGS1*. The empty vector served as control. Error bars represent standard deviations from three independent measurements. (G) Suppression of the temperature sensitivity of dna2Δ405N by overexpression of MGS1 requires catalytic activity, but not the UBZ domain. Overexpression was achieved by means of episomal (2 µ) vectors with the MGS1 promoter. Growth at 25°C and 37°C was monitored by spot assays. (H) Inactivation of the Mgs1 UBZ domain is sufficient to suppress the damage sensitivity of pol32 mutants. Sensitivity of the indicated strains to MMS was assessed by spot assays.

Mgs1 preferentially interacts with ubiquitylated PCNA in a UBZ-dependent manner. (A) Protein–protein interactions with full-length Mgs1 and the isolated UBZ domain (amino acids 1–47) were analysed in the two-hybrid system, using fusions to the Gal4 activation (AD) and DNA-binding (BD) domains. Interactions were tested with monoubiquitin (Ub*) and linear ubiquitin arrays (Ub*_n) either alone or fused to the N- or C-terminus of PCNA*. Asterisks indicate mutations in PCNA and ubiquitin that prevent further modification and processing by isopeptidases, and ‘L’ signifies a four-amino acid linker. Fusions of Ub* to GFP and of Smt3 (G98V, to prevent processing) to PCNA* were used as specificity controls. Empty vectors (–) served as negative controls. Presence of the constructs was confirmed by growth on medium lacking leucine and tryptophan (-LW), and positive signals were scored on medium additionally lacking histidine (-HLW) or—for strong interactions—histidine and adenine (-AHLW). (B) Interactions of Mgs1 with linear fusions of PCNA and ubiquitin were analysed in vitro. GST-tagged versions of Mgs1 and Mgs1* were immobilized on glutathione Sepharose and assayed for binding to the purified Ub*_n(L)–PCNA* constructs described in A, produced as His₆-tagged recombinant proteins, either in isolation or as an equimolar mixture. An anti-GST control blot is shown in Supplementary Figure S2B. (C) Interactions of Mgs1 were analysed in vitro as in B, but using PCNA subjected to in vitro mono- (M) or polyubiquitylation (P) at K164 prior to the assay. Unmodified PCNA that was either left unloaded (–) or loaded (+) onto DNA in parallel reactions served as controls. DNA was removed by nuclease treatment before performing the pull-down assays. Asterisks on the blot indicate cross-reactions of the anti-PCNA antibody with the GST fusion constructs. The bottom panel represents a weaker exposure of the blot.

Mgs1 negatively acts on Polymerase δ and PCNA. (A) Deletion of MGS1 or inactivation of its UBZ domain suppresses the damage sensitivity of pol3ΔCt. MMS sensitivities were determined by spot assays in the indicated strains. (B) Deletion of MGS1 or inactivation of its UBZ domain suppresses the temperature and damage sensitivity of a pol31 (hys2-1) mutant. Spot assays were performed as above either on MMS-containing plates or by incubation at the indicated temperatures. (C) Overexpression of MGS1 inhibits growth of the pol3ΔCt mutant in a UBZ-independent manner. Growth of the indicated cultures in rich medium at 30°C was recorded by following optical densities at 600 nm. (D) Overexpression of MGS1 inhibits growth of mutants with defective PCNA–Pol32 interactions in a UBZ-independent manner. Growth of the indicated strains was monitored by spot assays on rich medium at 30°C.