Identification of RFC(Ctf18p, Ctf8p, Dcc1p)

Mayer, Melanie L.; Gygi, Steven P.; Aebersold, Ruedi; Hieter, Philip

doi:10.1016/s1097-2765(01)00254-4

Cited by 291 publications

(165 citation statements)

References 58 publications

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“…mps3-3 cells held for 5 h at 37°C in nocodazole medium exhibited an even greater cohesion defect (23%), relative to identically treated wild type cells (3%). These levels of cohesion defects in mps3 mutants are similar to those exhibited by nonessential cohesion factors (trf4 at 20%, chl1 at 23%, ctf18 at 25%, ctf8 at 30%, and ctf18 at 35%) (12,13,15,16) and reveal that Mps3p indeed functions in cohesion. To further demonstrate that the increase in the number of GFP signals was due to precocious sister separation and not aneuploidy present at the beginning of the experiment, we quantified the number of GFP signals in G 1 cells arrested with ␣-factor.…”

Section: Mps3p Functions In Sister Chromatidsupporting

confidence: 63%

“…The cohesion defect (23 versus 3% after 5 h and 14.2 versus 2.75% after 3 h of nocodazole) for mps3 mutant cells, compared with wild type, is similar to levels exhibited by numerous nonessential cohesion factors, including trf4 (20%), chl1 (23%), ctf18 (25%), ctf8 (30%), and ctf18 (35%) (12,13,15,16). In contrast, mutations in essential cohesion factor genes, including ctf7, mcd1/scc1, irr1/ scc3, pds5, smc1, and smc3, typically exhibit twice this level (50 -60%) of sister separation (9, 21, 23, 32, 38 -41).…”

Section: Discussionmentioning

confidence: 68%

“…Cohesion Assay-Our modified cohesion assay strain was previously described (13,14,32). Briefly, Tet operator repeats integrated ϳ40 kb from the centromere of chromosome V are detected via binding of the stably integrated GFP-Tet repressor.…”

Section: Methodsmentioning

confidence: 99%

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The Spindle Pole Body Assembly Component Mps3p/Nep98p Functions in Sister Chromatid Cohesion

Antoniacci

Kenna

Uetz

et al. 2004

Journal of Biological Chemistry

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For successful chromosome segregation during mitosis, several processes must occur early in the cell cycle, including spindle pole duplication, DNA replication, and the establishment of cohesion between nascent sister chromatids. Spindle pole body duplication begins in G 1 and continues during early S-phase as spindle pole bodies mature and start to separate. Key steps in spindle pole body duplication are the sequential recruitment of Cdc31p and Spc42p by the nuclear envelope transmembrane protein Msp3p/Nep98p (herein termed Mps3p). Concurrent with DNA replication, Ctf7p/Eco1p (herein termed Ctf7p) ensures that nascent sister chromatids are paired together, identifying the products of replication as sister chromatids. Here, we provide the first evidence that the nuclear envelope spindle pole body assembly component Mps3p performs a function critical to sister chromatid cohesion. Mps3p was identified as interacting with Ctf7p from a genome-wide two-hybrid screen, and the physical interaction was confirmed by both in vivo (co-immunoprecipitation) and in vitro (GST pull-down) assays. An in vivo cohesion assay on new mps3/nep98 alleles revealed that loss of Mps3p results in precocious sister chromatid separation and that Mps3p functions after G 1 , coincident with Ctf7p. Mps3p is not required for cohesion during mitosis, revealing that Mps3p functions in cohesion establishment and not maintenance. Mutated Mps3p that results in cohesion defects no longer binds to Ctf7p in vitro, demonstrating that the interaction between Mps3p and Ctf7p is physiologically relevant. In support of this model, mps3 ctf7 double mutant cells exhibit conditional synthetic lethality. These findings document a new role for Mps3p in sister chromatid cohesion and provide novel insights into the mechanism by which a spindle pole body component, when mutated, contributes to aneuploidy.Microtubule-organizing center duplication, separation, and microtubule nucleation are all essential facets of proper chromosome segregation. In budding yeast, microtubule-organizing centers, called spindle pole bodies, function on both sides of the nuclear envelope to nucleate microtubules in both the cytoplasmic and nuclear volumes. Spindle pole body duplication is a multistep process that includes formation of a satellite, expansion into a cytoplasmic duplication plaque, half-bridge growth, and subsequent insertion into the nuclear envelope (1, 2). Evidence from genetic and electron microscopy studies indicates that spindle pole body duplication starts in G 1 and continues into early S-phase. Mps3p/Nep98p (herein termed Mps3p) is an essential nuclear envelope protein that is concentrated at the nuclear envelope half-bridge and required for spindle pole body assembly. Early in spindle pole body assembly, Mps3p functions to recruit and anchor Cdc31p to the half-bridge. Later, Mps3p is required for integration of Spc42p into the spindle pole duplication plaque (3, 4).Coincident with the latter portion of spindle pole body maturation and separation are the processes ...

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Section: Mps3p Functions In Sister Chromatidsupporting

confidence: 63%

Section: Discussionmentioning

confidence: 68%

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The Spindle Pole Body Assembly Component Mps3p/Nep98p Functions in Sister Chromatid Cohesion

Antoniacci

Kenna

Uetz

et al. 2004

Journal of Biological Chemistry

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“…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).…”

Section: Discussionmentioning

confidence: 99%

Requirement for ATP by the DNA Damage Checkpoint Clamp Loader

Majka

Chung

Burgers

2004

Journal of Biological Chemistry

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The DNA damage clamp loader replication factor C (RFC-Rad24) consists of the Rad24 protein and the four small Rfc2-5 subunits of RFC. This complex loads the heterotrimeric DNA damage clamp consisting of Rad17, Mec3, and Ddc1 (Rad17/3/1) onto partial duplex DNA in an ATP-dependent manner. Interactions between the clamp loader and the clamp have been proposed to mirror those of the replication clamp loader RFC and the sliding clamp proliferating cell nuclear antigen (PCNA). In that system, three ATP molecules bound to the Rfc2, Rfc3, and Rfc4 subunits are necessary and sufficient for efficient loading of PCNA, whereas ATP binding to Rfc1 is not required. In contrast, in this study, we show that mutant RFC-Rad24 with a rad24-K115E mutation in the ATP-binding domain of Rad24 shows defects in the ATPase of the complex and is defective for interaction with Rad17/3/1 and for loading of the checkpoint clamp. A similar defect was measured with a mutant RFCRad24 clamp loader carrying a rfc4K55R ATP-binding mutation, whereas the rfc4K55E clamp loader showed partial loading activity, in agreement with genetic studies of these mutants. These studies show that ATP utilization by the checkpoint clamp/clamp loader system is effectively different from that by the structurally analogous replication system.

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“…These facts may point to the existence of an alternative RFC complex, in which Rfc1p is replaced by Elg1p (26,27). At least two other similar RFC complexes exist in yeast; in these complexes, Rfc1p is replaced by either Ctf18p or Rad24p (28,29). Deletion of CTF18 causes telomere shortening (30) whereas a RAD24 deletion does not affect telomere length.…”

mentioning

confidence: 99%

ELG1 , a regulator of genome stability, has a role in telomere length regulation and in silencing

Smolikov

Mazor

Krauskopf

2004

Proc. Natl. Acad. Sci. U.S.A.

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Telomeres, the natural ends of eukaryotic chromosomes, prevent the loss of chromosomal sequences and preclude their recognition as broken DNA. Telomere length is kept under strict boundaries by the action of various proteins, some with negative and others with positive effects on telomere length. Recently, data have been accumulating to support a role for DNA replication in the control of telomere length, although through a currently poorly understood mechanism. Elg1p, a replication factor C (RFC)-like protein of yeast, contributes to genome stability through a putative replication-associated function. Here, we show that Elg1p participates in negative control of telomere length and in telomeric silencing through a replication-mediated pathway. We show that the telomeric function of Elg1 is independent of recombination and completely dependent on an active telomerase. Additionally, this function depends on yKu and DNA polymerase. We discuss alternative models to explain how Elg1p affects telomere length.DNA replication ͉ replication factor C ͉ Saccharomyces cerevisiae R eplication of a linear DNA molecule is carried out by DNA polymerases and additional proteins that duplicate the DNA by moving coordinately on both the lagging and leading strands (1). DNA attrition at the ends of chromosomes due to the ''end replication problem'' is avoided by the action of the specialized reverse transcriptase, called telomerase (2, 3). Telomerase adds G-rich DNA repeats termed telomeres onto the ends of chromosomes. In addition to their role in preventing loss of chromosomal sequences, telomeres serve as a boarding pad for a multiprotein complex that is implicated in several important cellular functions. Among these functions is capping telomeres, thus avoiding the recognition of the natural ends of chromosomes as broken DNA. This capping in turn prevents cell cycle arrest and chromosomal fusions, allowing proper chromosomal segregation (4).Many proteins, most of which are part of the telomeric complex, regulate telomere length in both positive and negative fashions (5). The best-studied pathway of telomerase-negative regulation is the Rap1p pathway in yeast. Rap1p is bound throughout the telomeric sequence and interacts with two proteins, Rif1p and Rif2p (6, 7). The resulting complex is thought to prevent telomerase from accessing the telomeric DNA substrate by a yet unknown mechanism. Additional factors were shown to be involved in telomere length regulation. Tel1p and the yKu70p-yKu80p heterodimer act as positive regulators of telomerase (8-10). Tel1p is proposed to act by recruiting the MRX (Mer11͞Rad50͞Xrs2) complex (11). The yKu70p͞yKu80p heterodimer influences telomere length by protecting telomere ends from degradation. It also has additional functions in telomeric silencing (through recruiting Sir proteins to the telomeres), telomeric nuclear localization, and telomere replication timing (12-15). Cdc13p binds the telomeric single strand, recruits telomerase to the telomere, and prevents degradation (16,17). By these fu...

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Identification of RFC(Ctf18p, Ctf8p, Dcc1p)

Cited by 291 publications

References 58 publications

The Spindle Pole Body Assembly Component Mps3p/Nep98p Functions in Sister Chromatid Cohesion

The Spindle Pole Body Assembly Component Mps3p/Nep98p Functions in Sister Chromatid Cohesion

Requirement for ATP by the DNA Damage Checkpoint Clamp Loader

ELG1 , a regulator of genome stability, has a role in telomere length regulation and in silencing

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