Mechanical Link between Cohesion Establishment and DNA Replication: Ctf7p/Eco1p, a Cohesion Establishment Factor, Associates with Three Different Replication Factor C Complexes

Abstract

CTF7/ECO1 is an essential yeast gene required for the establishment of sister chromatid cohesion. The findings that CTF7/ECO1, POL30 (PCNA), and CHL12/CTF18 (a replication factor C [RFC] homolog) genetically interact provided the first evidence that the processes of cohesion establishment and DNA replication are intimately coupled-a link now confirmed by other studies. To date, however, it is unknown how Ctf7p/Eco1p function is coupled to DNA replication or whether Ctf7p/Eco1p physically associates with any components of the DNA replication machinery. Here, we report that Ctf7p/Eco1p associates with proteins that perform partially redundant functions in DNA replication. Chl12p/Ctf18p combines with Rfc2p to Rfc5p to form one of three independent RFC complexes. By chromatographic methods, Ctf7p/Eco1p was found to associate with Chl12/Ctf18p and with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. The association between Ctf7p/Eco1p and this RFC complex is biologically relevant in that (i) Ctf7p/Eco1p cosediments with Chl12p/Ctf18p in vivo and (ii) rfc5-1 mutant cells exhibit precocious sister separation. Previous studies revealed that Rfc1p or Rad24p associates with Rfc2p to Rfc5p to form two other RFC complexes independent of Ctf18p-RFC complexes. These Rfc1p-RFC and Rad24p-RFC complexes function in DNA replication or repair and DNA damage checkpoint pathways. Importantly, Ctf7p/Eco1p also associates with Rfc1p and Rad24p, suggesting that these RFC complexes also play critical roles in cohesion establishment. The associations between Ctf7p/Eco1p and RFC subunits provide novel evidence regarding the physical linkage between cohesion establishment and DNA replication. Furthermore, the association of Ctf7p/Eco1p with each of three RFC complexes supplies new insights into the functional redundancy of RFC complexes in cohesion establishment.From the time of DNA replication until the onset of anaphase, sister chromatids remain tightly paired along their length (15,46,55). Cytological and molecular studies of cohesion factors reveal that this sister chromatid pairing, or cohesion, is an essential component of bipolar spindle formation, chromosome segregation, cell cycle progression, and doublestrand-break repair (25,49,50). Studies performed with numerous cell systems reveal that defects in cohesion between sister chromatids result in missegregation of both sisters to one daughter cell and cell death (16,23,31,35,51,52,57). In humans, chromosome missegregation allows for phenotypic expression of recessive mutations in tumor suppression and cancer-related growth control genes (29, 65).Three classes of proteins are required for sister chromatid cohesion and proper chromosome segregation. Structural cohesion proteins (or cohesins) provide the glue that maintains sister chromatid pairing from G 1 /S to anaphase onset. In budding yeast, the cohesins include Smc1p, Smc3p, Mcd1p/Scc1p, Scc3p/Irr1p, and Pds5p (16,19,28,35,42,52,57). Recent data revealed that the structural cohesins form a ring. This ring structure is thought to hold sister chr...

Requirement for ATP by the DNA Damage Checkpoint Clamp Loader

“…However, this simple argument ignores the complexity of interactions of the Rfc2-5 core, which can function in at least four distinct clamp loaders typified by RFC1, RAD24, CTF18, and ELG1 (23)(24)(25)(26)(27). Mutations in the latter two genes directly or indirectly affect DNA damage response and checkpoint function, and it is possible that the observed phenotypic defects of the RFC4 mutations carry additional contributions from possible defects in the function of the CTF18 and ELG1 clamp loaders (26 -28).…”

The Nuclear Envelope and Spindle Pole Body-Associated Mps3 Protein Bind Telomere Regulators and Function in Telomere Clustering

“…GST-CTF7 and GST-MPS3 expression in Escherichia coli cells and pull-downs from yeast lysate cells were performed as previously described. 13 Briefly, yeast cells containing Mps3p-13MYC, Mcd1p-MYC or Est1p-MYC (a generous gift of V. Zakian) were spheroplasted in 100T Zymolyase (Seikagaku), lysed by swelling and mechanical disruption (20 mM HEPES-HCl (pH 7.5), 5 mM MgCl 2 , and protease inhibitors) and centrifuged at 9500 rpm for 45 min (model JA-20; Beckman). The supernatant was removed, and the insoluble chromatin pellet extracted with lysis buffer containing 1 M NaCl before recentrifugation.…”

“…For instance, Mps3p is critical for proper sister chromatid cohesion and interacts both physically and genetically with the soluble cohesion establishment factor Ctf7p-an essential and highly conserved protein required for sister chromatid pairing during S-phase. [10][11][12][13][14] Based on these findings and previous reports that Ctf7p associates with DNA replication factors, 12,13,15 one model is that Mps3p recruits Ctf7p to the nuclear envelope-potentially activating Ctf7p before its release to the DNA replication fork to establish sister chromatid pairing. 10 Note that other spindle pole body components such as Kar3p and Bim1p were previously identified as functioning in sister chromatid cohesion.…”

MGO3 and GIP1 act synergistically for the maintenance of centromeric cohesion

“…1 In addition, the establishment of cohesion during S phase involves a chromosome transmission fidelity protein 7 (CTF7) which acetylates the structural maintenance of chromosome 3 (SMC3), a central component of the cohesion complex. 2 This step may also imply CTF18 and be coupled with the passage of the DNA replication fork. 2 This suggests that a mechanical link exists between cohesion establishment and DNA replication.…”

“…2 This step may also imply CTF18 and be coupled with the passage of the DNA replication fork. 2 This suggests that a mechanical link exists between cohesion establishment and DNA replication.…”