Replication Factor C-like Complex Research Articles

ATAD5 suppresses centrosome over-duplication by regulating UAF1 and ID1

ABSTRACT Centrosomes are the primary microtubule-organizing centers that are important for mitotic spindle assembly. Centrosome amplification is commonly observed in human cancer cells and contributes to genomic instability. However, it is not clear how centrosome duplication is dysregulated in cancer cells. Here, we report that ATAD5, a replisome protein that unloads PCNA from chromatin as a replication factor C-like complex (RLC), plays an important role in regulating centrosome duplication. ATAD5 is present at the centrosome, specifically at the base of the mother and daughter centrioles that undergo duplication. UAF1, which interacts with ATAD5 and regulates PCNA deubiquitination as a complex with ubiquitin-specific protease 1, is also localized at the centrosome. Depletion of ATAD5 or UAF1 increases cells with over-duplicated centrosome whereas ATAD5 overexpression reduces such cells. Consistently, the proportion of cells showing the multipolar mode of chromosome segregation is increased among ATAD5-depleted cells. The localization and function of ATAD5 at the centrosomes do not require other RLC subunits. UAF1 interacts and co-localizes with ID1, a protein that increases centrosome amplification upon overexpression. ATAD5 depletion reduces interactions between UAF1 and ID1 and increases ID1 signal at the centrosome, providing a mechanistic framework for understanding the role of ATAD5 in centrosome duplication.

CHTF18 ensures the quantity and quality of the ovarian reserve†.

The number and quality of oocytes, as well as the decline in both of these parameters with age, determines reproductive potential in women. However, the underlying mechanisms of this diminution are incompletely understood. Previously, we identified novel roles for CHTF18 (Chromosome Transmission Fidelity Factor 18), a component of the conserved Replication Factor C-like complex, in male fertility and gametogenesis. Currently, we reveal crucial roles for CHTF18 in female meiosis and oocyte development. Chtf18-/- female mice are subfertile and have fewer offspring beginning at 6months of age. Consistent with age-dependent subfertility, Chtf18-/- ovaries contain fewer follicles at all stages of folliculogenesis than wild type ovaries, but the decreases are more significant at 3 and 6 months of age. By 6 months of age, both primordial and growing ovarian follicle pools are markedly reduced to near depletion. Chromosomal synapsis in Chtf18-/- oocytes is complete, but meiotic recombination is impaired resulting in persistent DNA double-strand breaks, fewer crossovers, and early homolog disjunction during meiosis I. Consistent with poor oocyte quality, the majority of Chtf18-/- oocytes fail to progress to metaphase II following meiotic resumption and a significant percentage of those that do progress are aneuploid. Collectively, our findings indicate critical functions for CHTF18 in ensuring both the quantity and quality of the mammalian oocyte pool.

Identification of Elg1 interaction partners and effects on post-replication chromatin re-formation.

Elg1, the major subunit of a Replication Factor C-like complex, is critical to ensure genomic stability during DNA replication, and is implicated in controlling chromatin structure. We investigated the consequences of Elg1 loss for the dynamics of chromatin re-formation following DNA replication. Measurement of Okazaki fragment length and the micrococcal nuclease sensitivity of newly replicated DNA revealed a defect in nucleosome organization in the absence of Elg1. Using a proteomic approach to identify Elg1 binding partners, we discovered that Elg1 interacts with Rtt106, a histone chaperone implicated in replication-coupled nucleosome assembly that also regulates transcription. A central role for Elg1 is the unloading of PCNA from chromatin following DNA replication, so we examined the relative importance of Rtt106 and PCNA unloading for chromatin reassembly following DNA replication. We find that the major cause of the chromatin organization defects of an ELG1 mutant is PCNA retention on DNA following replication, with Rtt106-Elg1 interaction potentially playing a contributory role.

Down-regulate of Djrfc2 causes tissues hypertrophy during planarian regeneration

Planarians are an ideal model organism for regeneration research due to their amazing ability to regenerate. DNA replication is crucial for genome stability. Replication factor C (RFC), which is a replication factor C-like complex and plays an important role during DNA replication in eukaryotes, has been reported as a wound response factor during planarian regeneration. However, how RFC controls regeneration in planarians by regulating DNA replication remains to be explained. Here, we used a two-dimensional electrophoresis (2-DE) proteomic approach to identify differentially expressed proteins in intact and regenerated planarians. Approximately 132 protein spots showed differences between intact and regenerative tissues. We selected 21 significantly expressed protein spots and processed them using TOF MS analysis. Finally, we cloned three of these candidate genes (Djhsp70, Djrfc2, Djfaim), focusing on the function of Djrfc2 during regeneration. We found that the distribution of Djrfc2 tends toward the wound site. RNA interference (RNAi) of Djrfc2 increases the number of dividing cells and the expression level of planarian neoblast marker genes, which may result in hyper-proliferation. Our studies use an available approach to directly study the regeneration dynamic at the protein level and provide further evidence to support a function of Djrfc2 in planarian regeneration.

A New Method, "Reverse Yeast Two-Hybrid Array" (RYTHA), Identifies Mutants that Dissociate the Physical Interaction Between Elg1 and Slx5.

The vast majority of processes within the cell are carried out by proteins working in conjunction. The Yeast Two-Hybrid (Y2H) methodology allows the detection of physical interactions between any two interacting proteins. Here, we describe a novel systematic genetic methodology, "Reverse Yeast Two-Hybrid Array" (RYTHA), that allows the identification of proteins required for modulating the physical interaction between two given proteins. Our assay starts with a yeast strain in which the physical interaction of interest can be detected by growth on media lacking histidine, in the context of the Y2H methodology. By combining the synthetic genetic array technology, we can systematically screen mutant libraries of the yeast Saccharomyces cerevisiae to identify trans-acting mutations that disrupt the physical interaction of interest. We apply this novel method in a screen for mutants that disrupt the interaction between the N-terminus of Elg1 and the Slx5 protein. Elg1 is part of an alternative replication factor C-like complex that unloads PCNA during DNA replication and repair. Slx5 forms, together with Slx8, a SUMO-targeted ubiquitin ligase (STUbL) believed to send proteins to degradation. Our results show that the interaction requires both the STUbL activity and the PCNA unloading by Elg1, and identify topoisomerase I DNA-protein cross-links as a major factor in separating the two activities. Thus, we demonstrate that RYTHA can be applied to gain insights about particular pathways in yeast, by uncovering the connection between the proteasomal ubiquitin-dependent degradation pathway, DNA replication, and repair machinery, which can be separated by the topoisomerase-mediated cross-links to DNA.

PCNA Retention on DNA into G2/M Phase Causes Genome Instability in Cells Lacking Elg1

SummaryLoss of the genome maintenance factor Elg1 causes serious genome instability that leads to cancer, but the underlying mechanism is unknown. Elg1 forms the major subunit of a replication factor C-like complex, Elg1-RLC, which unloads the ring-shaped polymerase clamp PCNA from DNA during replication. Here, we show that prolonged retention of PCNA on DNA into G2/M phase is the major cause of genome instability in elg1Δ yeast. Overexpression-induced accumulation of PCNA on DNA causes genome instability. Conversely, disassembly-prone PCNA mutants that relieve PCNA accumulation rescue the genome instability of elg1Δ cells. Covalent modifications to the retained PCNA make only a minor contribution to elg1Δ genome instability. By engineering cell-cycle-regulated ELG1 alleles, we show that abnormal accumulation of PCNA on DNA during S phase causes moderate genome instability and its retention through G2/M phase exacerbates genome instability. Our results reveal that PCNA unloading by Elg1-RLC is critical for genome maintenance.

Replication-Coupled PCNA Unloading by the Elg1 Complex Occurs Genome-wide and Requires Okazaki Fragment Ligation.

SummaryThe sliding clamp PCNA is a crucial component of the DNA replication machinery. Timely PCNA loading and unloading are central for genome integrity and must be strictly coordinated with other DNA processing steps during replication. Here, we show that the S. cerevisiae Elg1 replication factor C-like complex (Elg1-RLC) unloads PCNA genome-wide following Okazaki fragment ligation. In the absence of Elg1, PCNA is retained on chromosomes in the wake of replication forks, rather than at specific sites. Degradation of the Okazaki fragment ligase Cdc9 leads to PCNA accumulation on chromatin, similar to the accumulation caused by lack of Elg1. We demonstrate that Okazaki fragment ligation is the critical prerequisite for PCNA unloading, since Chlorella virus DNA ligase can substitute for Cdc9 in yeast and simultaneously promotes PCNA unloading. Our results suggest that Elg1-RLC acts as a general PCNA unloader and is dependent upon DNA ligation during chromosome replication.

The Elg1 Replication Factor C-like Complex Functions in PCNA Unloading during DNA Replication

The ring-shaped complex PCNA coordinates DNA replication, encircling DNA to act as a polymerase clamp and a sliding platform to recruit other replication proteins. PCNA is loaded onto DNA by replication factor C, but it has been unknown how PCNA is removed from DNA when Okazaki fragments are completed or the replication fork terminates. Here we show that the Elg1 replication factor C-like complex (Elg1-RLC) functions in PCNA unloading. Using an improved degron system we show that without Elg1, PCNA accumulates on Saccharomyces cerevisiae chromatin during replication. The accumulated PCNA can be removed from chromatin in vivo by switching on Elg1 expression. We find moreover that treating chromatin with purified Elg1-RLC causes PCNA unloading in vitro. Our results demonstrate that Elg1-RLC functions in unloading of both unmodified and SUMOylated PCNA during DNA replication, while the genome instability of an elg1Δ mutant suggests timely PCNA unloading is critical for chromosome maintenance.

Disruption of Chtf18 Causes Defective Meiotic Recombination in Male Mice

CHTF18 (chromosome transmission fidelity factor 18) is an evolutionarily conserved subunit of the Replication Factor C-like complex, CTF18-RLC. CHTF18 is necessary for the faithful passage of chromosomes from one daughter cell to the next during mitosis in yeast, and it is crucial for germline development in the fruitfly. Previously, we showed that mouse Chtf18 is expressed throughout the germline, suggesting a role for CHTF18 in mammalian gametogenesis. To determine the role of CHTF18 in mammalian germ cell development, we derived mice carrying null and conditional mutations in the Chtf18 gene. Chtf18-null males exhibit 5-fold decreased sperm concentrations compared to wild-type controls, resulting in subfertility. Loss of Chtf18 results in impaired spermatogenesis; spermatogenic cells display abnormal morphology, and the stereotypical arrangement of cells within seminiferous tubules is perturbed. Meiotic recombination is defective and homologous chromosomes separate prematurely during prophase I. Repair of DNA double-strand breaks is delayed and incomplete; both RAD51 and γH2AX persist in prophase I. In addition, MLH1 foci are decreased in pachynema. These findings demonstrate essential roles for CHTF18 in mammalian spermatogenesis and meiosis, and suggest that CHTF18 may function during the double-strand break repair pathway to promote the formation of crossovers.

Quantitative Proteomic Analysis of Chromatin Reveals that Ctf18 Acts in the DNA Replication Checkpoint

Yeast cells lacking Ctf18, the major subunit of an alternative Replication Factor C complex, have multiple problems with genome stability. To understand the in vivo function of the Ctf18 complex, we analyzed chromatin composition in a ctf18Δ mutant using the quantitative proteomic technique of stable isotope labeling by amino acids in cell culture. Three hundred and seven of the 491 reported chromosomal proteins were quantitated. The most marked abnormalities occurred when cells were challenged with the replication inhibitor hydroxyurea. Compared with wild type, hydroxyurea-treated ctf18Δ cells exhibited increased chromatin association of replisome progression complex components including Cdc45, Ctf4, and GINS complex subunits, the polymerase processivity clamp PCNA and the single-stranded DNA-binding complex RPA. Chromatin composition abnormalities observed in ctf18Δ cells were very similar to those of an mrc1Δ mutant, which is defective in the activating the Rad53 checkpoint kinase in response to DNA replication stress. We found that ctf18Δ cells are also defective in Rad53 activation, revealing that the Ctf18 complex is required for engagement of the DNA replication checkpoint. Inappropriate initiation of replication at late origins, because of loss of the checkpoint, probably causes the elevated level of chromatin-bound replisome proteins in the ctf18Δ mutant. The role of Ctf18 in checkpoint activation is not shared by all Replication Factor C-like complexes, because proteomic analysis revealed that cells lacking Elg1 (the major subunit of a different Replication Factor C-like complex) display a different spectrum of chromatin abnormalities. Identification of Ctf18 as a checkpoint protein highlights the usefulness of chromatin proteomic analysis for understanding the in vivo function of proteins that mediate chromatin transactions.

Identification of protein complexes required for efficient sister chromatid cohesion.

Ctf8p is a component of Ctf18-RFC, an alternative replication factor C-like complex required for efficient sister chromatid cohesion in Saccharomyces cerevisiae. We performed synthetic genetic array (SGA) analysis with a ctf8 deletion strain as a primary screen to identify other nonessential genes required for efficient sister chromatid cohesion. We then assessed proficiency of cohesion at three chromosomal loci in strains containing deletions of the genes identified in the ctf8 SGA screen. Deletion of seven genes (CHL1, CSM3, BIM1, KAR3, TOF1, CTF4, and VIK1) resulted in defective sister chromatid cohesion. Mass spectrometric analysis of immunoprecipitated complexes identified a physical association between Kar3p and Vik1p and an interaction between Csm3p and Tof1p that we confirmed by coimmunoprecipitation from cell extracts. These data indicate that synthetic genetic array analysis coupled with specific secondary screens can effectively identify protein complexes functionally related to a reference gene. Furthermore, we find that genes involved in mitotic spindle integrity and positioning have a previously unrecognized role in sister chromatid cohesion.