Physical links between the nuclear envelope protein Mps3, three alternate replication factor C complexes, and a variant histone in Saccharomyces cerevisiae

Abstract

Viability of cell progeny upon cell division require that genomes are replicated, repaired, and maintained with high fidelity. Central to both DNA replication and repair are Replication Factor C (RFC) complexes which catalyze the unloading/loading of sliding clamps such as PCNA or 9-1-1 complexes on DNA. Budding yeast contain four alternate RFC complexes which play partially redundant roles. Rfc1, Ctf18, Rad24, and Elg1 are all large subunits that bind, in a mutually exclusive fashion to RFC 2-5 small subunits. Ctf18, Rad24, and Elg1 are of particular interest because, in addition to their roles in maintaining genome integrity, all three play critical roles in sister chromatid tethering reactions that appear coupled to their roles in DNA replication/repair. Intriguingly, the nuclear envelope protein Mps3 similarly exhibits roles in repair and cohesion, leading us to hypothesize that Mps3 and RFCs function through a singular mechanism. Here we report that the nuclear envelope protein Mps3 physically associates with all three of these large RFC complex subunits (Ctf18, Elg1, and Rad24). In addition we report a physical interaction between Mps3 and the histone variant Htz1, a factor previously shown to promote DNA repair. In combination, these findings reveal a direct link between the nuclear envelope and chromatin and provide support for a model that telomeres and chromatin interact with the nuclear envelope during both DNA repair and sister chromatid pairing reactions.

FIG. 1.

Ctf18 physically associates with Mps3. Lane 1: Ctf18-MYC S2 beads coupled with GST-Mps3, Lane 2: Ctf18-MYC S2 with beads, Lane 3: Ctf18-MYC S2 with beads coupled with GST, Lane 4: S2 extract from cells expressing Ctf18-MYC. GST-Mps3 or GST expression was induced in E. coli cells using IPTG. S2 extracts of Ctf18-MYC (YJH40.4) were prepared according to Kalinich and Douglas . The supernatants were then harvested and divided into three equal aliquots. The three aliquots were incubated with one of the three bead matrices (glutathione-Sepharose beads, beads coupled to GST, or beads coupled to GST-Mps3). Incubations were performed at 4°C for 2 h. The treated beads were washed several times before bound proteins were removed using SDS-containing solubilization buffer. Samples were analyzed by Western blot using rabbit antibodies raised against MYC to show the presence of Ctf18-MYC in these extracts.

FIG. 2.

Rad24 physically interacts with Mps3. Lane 1: Rad24-FLAG S1 with glutathione-Sepharose beads coupled to GST-Mps3, Lane 2: Rad24-FLAG S1 with beads coupled with GST, Lane 3: Rad24-FLAG S1 with beads, Lane 4: Rad24-FLAG S2 with glutathione sepharose beads coupled to GST-Mps3, Lane 5: Rad24-FLAG S2 with beads coupled to GST, Lane 6: Rad24-FLAG S2 with beads, Lane 7: Extract from cells expressing Rad24-FLAG. GST-Mps3 or GST expression was induced in E. coli cells using IPTG. S1 and S2 extracts of Rad24-FLAG (KSC1377) were prepared according to Kalinich and Douglas . The supernatants were then harvested and divided into three equal aliquots. The three aliquots were incubated with one of the three bead matrices (glutathione-Sepharose beads, beads coupled to GST, or GST-Mps3). Incubations were performed at 4°C for 2 h. The treated beads were washed several times before bound proteins were removed using SDS-containing solubilization buffer. Samples were analyzed by Western blot using monoclonal antibodies raised against FLAG to show the presence of Rad24-FLAG in these extracts.

FIG. 3.

Elg1 physically associates with Mps3. Lane 1: S2 extract from cells expressing Elg1-MYC, Lane 2: Elg1-MYC S2 with beads, Lane 3: Elg1-MYC S2 with beads coupled with GST, Lane 4: Elg1-MYC S2 beads coupled with GST-Mps3. GST-Mps3 or GST expression was induced in E. coli cells using IPTG. S2 extracts of Elg1-MYC (YDD282) were prepared according to Kalinich and Douglas . The supernatants were then harvested and divided into three equal aliquots. The three aliquots were incubated with one of the three bead matrices (glutathione-Sepharose beads, beads coupled to GST, or beads coupled to GST-Mps3). Incubations were performed at 4°C for 2 h. The treated beads were washed several times before bound proteins were removed using SDS-containing solubilization buffer. Samples were analyzed by Western blot using rabbit antibodies raised against MYC to show the presence of Elg1-MYC in these extracts.

FIG. 4.

MPS3 physically associates with Htz1 in vivo. (A) Lane 1: Cell lysates expressing Mps3-MYC and Htz1 incubated without (−) MYC antibody and then precipitated with Protein A-sepharose beads, Lane 2: Cell lysate expressing Mps3-MYC and Htz1, Lane 3: Cell lysates expressing Mps3-MYC and Htz1 incubated with (+) MYC antibody and then precipitated with Protein A-sepharose beads (B) Lane1 and Lane 2: Cell lysates expressing Mps3-MYC and Htz1 incubated without (−) or with (+) MYC antibody and then precipitated with Protein A-sepharose beads. Lane 3 and Lane 4: Cell lysates expressing mps3-3-MYC and Htz1 incubated without (−) or with (+) MYC antibody and then precipitated with Protein A-sepharose beads. Western blot analysis using antibodies against Htz1 reveal that both Mps3 and mps3-3 co-immunoprecipitate with Htz1p.

FIG. 5.

Conditional lethality of mps3-3/htz1Δ strain. Wild type, single mutant mps3-3, knockout htz1Δ, and double mutant htz1Δ mps3-3 were plated on YPD medium in 10-fold serial dilutions and placed at 23°C, 33°C, and 37°C. All strains grew at 23°C. At 33°C wild type and both single mutants are viable. The htz1Δ mps3-3 double mutant growth is severely retarded at 33°C compared with wild type and both single mutants revealing a conditionally synthetic lethal genetic interaction between the two genes. The mps3-3 single mutant and htz1Δ mps3-3 double mutant were inviable at 37°C as expected due to mps3-3 temperature sensitivity.

FIG. 6.

Htz1 and Mps3 function in DNA damage response/repair. Wild type, mps3-3, htz1Δ, and mps3-3 htz1Δ were grown in 10-fold serial dilutions at 23°C on YPD rich medium, 30 mM HU, or 0.01% MMS. HU, hydroxyurea; MMS, methyl methanesulfonate.