Rtt101 and Mms1 in budding yeast form a CUL4(DDB1)-like ubiquitin ligase that promotes replication through damaged DNA

Abstract

In budding yeast the cullin Rtt101 promotes replication fork progression through natural pause sites and areas of DNA damage, but its relevant subunits and molecular mechanism remain poorly understood. Here, we show that in budding yeast Mms1 and Mms22 are functional subunits of an Rtt101-based ubiquitin ligase that associates with the conjugating-enzyme Cdc34. Replication forks in mms1Delta, mms22Delta and rtt101Delta cells are sensitive to collisions with drug-induced DNA lesions, but not to transient pausing induced by nucleotide depletion. Interaction studies and sequence analysis have shown that Mms1 resembles human DDB1, suggesting that Rtt101(Mms1) is the budding yeast counterpart of the mammalian CUL4(DDB1) ubiquitin ligase family. Rtt101 interacts in an Mms1-dependent manner with the putative substrate-specific adaptors Mms22 and Crt10, the latter being a regulator of expression of ribonucleotide reductase. Taken together, our data suggest that the Rtt101(Mms1) ubiquitin ligase complex might be required to reorganize replication forks that encounter DNA lesions.

Figure 1

RTT101 interacts with MMS1, MMS22 and CDC34. (A) The indicated yeast deletion mutants were grown overnight to saturation in YPD medium at 30°C and serially diluted onto YPD plates either in the presence (right panel) or absence (left panel) of 0.0025% MMS, and incubated for a further 2 days at 30°C. (B) MMS22, MMS1 and RTT101 interact genetically with many of the same genes involved in DNA replication and homologous recombination. Green, synthetic lethality; grey, synthetic growth defect. (C) rtt101Δ and cdc34-2 mutants were grown overnight in YPD medium at 25°C, and serial dilutions were spotted onto YPD media containing, as indicated, 0.01% MMS or 10 μM CPT. Cells were grown for 2 days at 32.5°C. (D) Bacterially expressed GST, GST-Rad6, GST-Ubc4 and GST-Cdc34 were immobilized onto glutathione beads and incubated with yeast extracts containing HA-Rtt101. Bound HA-Rtt101 was detected by Western blotting (upper panel), whereas the purified GST fusion proteins were visualized by Ponceau S staining (lower panel, the band corresponding to full-length GST-Cdc34 is marked with an asterisk). CPT, camptothecin; GST, glutathione S-transferase; HA, haemagglutinin; MMS, methyl methanesulphonate; YPD, yeast extract, peptone, dextrose; WT, wild type.

Figure 2

mms1Δ, mms22Δ and rtt101Δ cells are unable to replicate past MMS-induced DNA damage. (A) Wild-type, rtt101Δ, mms1Δ and mms22Δ cells were arrested in G₁ with α-factor and released into S phase in the presence of 0.033% MMS and 400 μg/ml BrdU. After 60 min, the cells were resuspended in a fresh medium containing BrdU. The persistence of unreplicated gaps along individual chromosomes was monitored by DNA combing 130 min after release from MMS. Representative DNA fibres are shown, with arrowheads pointing to unreplicated gaps. Green: BrdU; red: DNA. Scale bar, 50 kb. (B) The frequency of unreplicated gaps in wild-type, rtt101Δ, mms1Δ and mms22Δ cells was quantified 130 min after release from MMS. (C) The indicated yeast strains expressing endogenously tagged Rad53-TAP were grown to exponential phase at 30°C in YPD (0−) after which MMS was added for 2 h (0.02% final concentration). MMS was then quenched with sodium thiosulphate and the cells were released into pre-warmed YPD media (0+). Whole-cell protein extracts prepared at the indicated time points were probed for Rad53-TAP by Western blotting with the PAP antibody (Sigma, Buchs, Switzerland). (D) The indicated yeast mutants expressing 6 × HIS-tagged PCNA were grown and exposed to MMS as outlined in (C). PCNA was precipitated in the presence of 6 M guanidine HCl to preserve sensitive post-translational modifications, and ubiquitylated PCNA was detected using mouse monoclonal antibodies directed against ubiquitin (Cell Signaling, Allschwil, Switzerland). BrdU, 5-bromodeoxyuridine; HU, hydroxyurea; MMS, methyl methanesulphonate; PAP, peroxidase anti-peroxidase; PCNA, proliferating-cell nuclear antigen; YPD, yeast extract, peptone, dextrose; WT, wild type.

Figure 3

Mms1 belongs to the DDB1 family of cullin 4 adaptors. (A) Extracts prepared from wild-type (−), crt10Δ and mms22Δ cells expressing protein A-tagged Mms1 and either full-length (WT) or amino-terminally truncated (ΔN50) CBP-9Myc-Rtt101 were incubated with calmodulin beads, and the total extract (upper panels) and eluate (lower panels) were probed for the presence of bound Rtt101 and Mms1. (B) Three-dimensional representation of the DDB1 structure adapted from Li et al (2006); PDB accession 2b5m. The three β-propeller domains are labelled as BPA, BPB and BPC. Each propeller is characterized by seven repeats of antiparallel four-stranded β-sheets and can be assigned to distinct functions. BPB has been described to be a cullin-binding domain, whereas the BPA and BPC domains seem to bind to substrates. Stretches found to be conserved between Mms1 and DDB1 homologues are coloured in orange. (C) Full-length and the indicated N-terminal Rtt101 truncation mutants Δ50 and Δ100 were expressed as the only Rtt101 copy from GAL1,10 promoter. An empty vector (Vc) acted as a negative control. (D) Dendrogram of the DDB1/Mms1 homology using the neighbour-joining algorithm. For the tree construction, only reliable and gap-free alignment columns were used. Species names: Ag, Anopheles gambiae; At, Arabidopsis thaliana; Br, Brachydanio rerio; Ca, Candida albicans; Dm, Drosophila melanogaster; Eg, Eremothecium gossypii; En, Emericella nidulans; Hs, Homo sapiens; Kw, Kluyveromyces waltii; Mm, Mus musculus; Nc, Neurospora crassa; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Xt, Xenopus tropicalis. The principal subfamilies are indicated at the right border of the figure. BPA, β-propeller A; BPB, β-propeller B; BPC, β-propeller C; DDB1, DNA-damage-binding protein 1; PDB, Protein Data Bank.

Figure 4

Mms1 bridges the interaction of Mms22 and Crt10 with Rtt101. (A,B) Extracts prepared from wild-type (−) and mms1Δ cells expressing, as indicated, either full-length (WT) or N-terminally truncated (ΔN50) CBP-9Myc-Rtt101 together with protein A-tagged Crt10 (A) or Mms22 (B) were incubated with calmodulin beads. The total extract (upper panels) and eluates (lower panels) were then probed for the presence of bound Rtt101, Crt10 and Mms22. In (B), the extracts were prepared from cells treated for 20 min with or without 0.1% MMS. Note that the interaction of Rtt101 with Mms22 is regulated by DNA damage. (C) The DDB1-like protein Mms1 interacts with the N-terminal domain of Rtt101 and allows the recruitment of the putative substrate-specific adaptors Crt10 and Mms22. The Rtt101^Mms1/Crt10 complex might regulate the activity of RNR, whereas the Rtt101^Mms1/Mms22 complex promotes replication through DNA lesions, most likely through ubiquitination of an unknown target protein. DDB1, DNA-damage-binding protein 1; MMS, methyl methanesulphonate; RNR, ribonucleotide reductase.