Vertebrate CTF18 and DDX11 essential function in cohesion is bypassed by preventing WAPL-mediated cohesin release.

The alternative PCNA loader containing CTF18-DCC1-CTF8 facilitates sister chromatid cohesion (SCC) by poorly defined mechanisms. Here we found that in DT40 cells, CTF18 acts complementarily with the Warsaw breakage syndrome DDX11 helicase in mediating SCC and proliferation. We uncover that the lethality and cohesion defects of ctf18 ddx11 mutants are associated with reduced levels of chromatin-bound cohesin and rescued by depletion of WAPL, a cohesin-removal factor. On the contrary, high levels of ESCO1/2 acetyltransferases that acetylate cohesin to establish SCC do not rescue ctf18 ddx11 phenotypes. Notably, the tight proximity of sister centromeres and increased anaphase bridges characteristic of WAPL-depleted cells are abrogated by loss of both CTF18 and DDX11 The results reveal that vertebrate CTF18 and DDX11 collaborate to provide sufficient amounts of chromatin-loaded cohesin available for SCC generation in the presence of WAPL-mediated cohesin-unloading activity. This process modulates chromosome structure and is essential for cellular proliferation in vertebrates.

Division of Labor between PCNA Loaders in DNA Replication and Sister Chromatid Cohesion Establishment.

Concomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Several replication-fork-associated "cohesion establishment factors," including the multifunctional Ctf18-RFC complex, aid this process in as yet unknown ways. Here, we show that Ctf18-RFC's role in sister chromatid cohesion correlates with PCNA loading but is separable from its role in the replication checkpoint. Ctf18-RFC loads PCNA with a slight preference for the leading strand, which is dispensable for DNA replication. Conversely, the canonical Rfc1-RFC complex preferentially loads PCNA onto the lagging strand, which is crucial for DNA replication but dispensable for sister chromatid cohesion. The downstream effector of Ctf18-RFC is cohesin acetylation, which we place toward a late step during replication maturation. Our results suggest that Ctf18-RFC enriches and balances PCNA levels at the replication fork, beyond the needs of DNA replication, to promote establishment of sister chromatid cohesion and possibly other post-replicative processes.

Cryo-EM reveals a nearly complete PCNA loading process and unique features of the human alternative clamp loader CTF18-RFC.

The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded onto DNA by a clamp loader, e.g., the well-studied pentameric ATPase complex RFC (RFC1-5). The CTF18-RFC complex is an alternative clamp loader found recently to bind the leading strand DNA polymerase ε and load PCNA onto leading strand DNA, but its structure and the loading mechanism have been unknown. By cryo-EM analysis of in vitro assembled human CTF18-RFC-DNA-PCNA complex, we have captured seven loading intermediates, revealing a detailed PCNA loading mechanism onto a 3'-ss/dsDNA junction by CTF18-RFC. Interestingly, the alternative loader has evolved a highly mobile CTF18 AAA+ module likely to lower the loading activity, perhaps to avoid competition with the RFC and to limit its role to leading strand clamp loading. To compensate for the lost stability due to the mobile AAA+ module, CTF18 has evolved a unique ß-hairpin motif that reaches across RFC2 to interact with RFC5, thereby stabilizing the pentameric complex. Further, we found that CTF18 also contains a separation pin to locally melt DNA from the 3'-end of the primer; this ensures its ability to load PCNA to any 3'-ss/dsDNA junction, facilitated by the binding energy of the E-plug to the major groove. Our study reveals unique structural features of the human CTF18-RFC and contributes to a broader understanding of PCNA loading by the alternative clamp loaders.

Multiple roles of Pol epsilon in eukaryotic chromosome replication.

Pol epsilon is a tetrameric assembly that plays distinct roles during eukaryotic chromosome replication. It catalyses leading strand DNA synthesis; yet this function is dispensable for viability. Its non-catalytic domains instead play an essential role in the assembly of the active replicative helicase and origin activation, while non-essential histone-fold subunits serve a critical function in parental histone redeposition onto newly synthesised DNA. Furthermore, Pol epsilon plays a structural role in linking the RFC-Ctf18 clamp loader to the replisome, supporting processive DNA synthesis, DNA damage response signalling as well as sister chromatid cohesion. In this minireview, we discuss recent biochemical and structural work that begins to explain various aspects of eukaryotic chromosome replication, with a focus on the multiple roles of Pol epsilon in this process.

Histones on fire: the effect of Dun1 and Mrc1 on origin firing and replication of hyper-acetylated genomes.

As cells replicate their DNA, there is a need to synthesize new histones with which to wrap it. Newly synthesized H3 histones that are incorporated into the assembling chromatin behind the replication fork are acetylated at lysine 56. The acetylation is removed by two deacetylases, Hst3 and Hst4. This process is tightly regulated and any perturbation leads to genomic instability and replicative stress. We recently showed that Dun1, a kinase implicated mainly in the regulation of dNTPs, is vital in cells with hyper-acetylation, to counteract Rad53's inhibition on late-firing origins of replication. Our work showed that ∆hst3 ∆hst4 cells depend on late origin firing for survival, and are unable to prevent Rad53's inhibition when Dun1 is inactive. Thus, our work describes a role for Dun1 that is independent on its known function as a regulator of dNTP levels. Here we show that Mrc1 (Claspin in mammals), a protein that moves with the replicating fork and participates in both replication and checkpoint functions, plays also an essential role in the absence of H3K56Ac deacetylation. The sum of the results shown here and in our recent publication suggests that dormant origins are also utilized in these cells, making Mrc1, which regulates firing from these origins, also essential when histone H3 is hyper-acetylated. Thus, cells suffering from hyper-acetylation of H3K56 experience replication stress caused by a combination of prone-to-collapse forks and limited replication tracts. This combination makes both Dun1 and Mrc1, each acting on different targets, essential for viability.

Structural studies of RFCCtf18 reveal a novel chromatin recruitment role for Dcc1.

Replication factor C complexes load and unload processivity clamps from DNA and are involved in multiple DNA replication and repair pathways. The RFCCtf18 variant complex is required for activation of the intra-S-phase checkpoint at stalled replication forks and aids the establishment of sister chromatid cohesion. Unlike other RFC complexes, RFCCtf18 contains two non-Rfc subunits, Dcc1 and Ctf8. Here, we present the crystal structure of the Dcc1-Ctf8 heterodimer bound to the C-terminus of Ctf18. We find that the C-terminus of Dcc1 contains three-winged helix domains, which bind to both ssDNA and dsDNA We further show that these domains are required for full recruitment of the complex to chromatin, and correct activation of the replication checkpoint. These findings provide the first structural data on a eukaryotic seven-subunit clamp loader and define a new biochemical activity for Dcc1.

Alternative clamp loaders/unloaders.

Each time a cell duplicates, the whole genome must be accurately copied and distributed. The enormous amount of DNA in eukaryotic cells requires a high level of coordination between polymerases and other DNA and chromatin-interacting proteins to ensure timely and accurate DNA replication and chromatin formation. PCNA forms a ring that encircles the DNA. It serves as a processivity factor for DNA polymerases and as a landing platform for different proteins that interact with DNA and chromatin. It thus serves as a signaling hub and influences the rate and accuracy of DNA replication, the r-formation of chromatin in the wake of the moving fork and the proper segregation of the sister chromatids. Four different, conserved, protein complexes are in charge of loading/unloading PCNA and similar molecules onto DNA. Replication factor C (RFC) is the canonical complex in charge of loading PCNA, the replication clamp, during S-phase. The Rad24, Ctf18 and Elg1 proteins form complexes similar to RFC, with particular functions in the cell's nucleus. Here we summarize our current knowledge about the roles of these important factors in yeast.

CTF18-RFC contributes to cellular tolerance against chain-terminating nucleoside analogs (CTNAs) in cooperation with proofreading exonuclease activity of DNA polymerase ε.

Chemotherapeutic nucleoside analogs, such as cytarabine (Ara-C), are incorporated into genomic DNA during replication. Incorporated Ara-CMP (Ara-cytidine monophosphate) serves as a chain terminator and inhibits DNA synthesis by replicative polymerase epsilon (Polε). The proofreading exonuclease activity of Polε removes the misincorporated Ara-CMP, thereby contributing to the cellular tolerance to Ara-C. Purified Polε performs proofreading, and it is generally believed that proofreading in vivo does not need additional factors. In this study, we demonstrated that the proofreading by Polε in vivo requires CTF18, a component of the leading-strand replisome. We found that loss of CTF18 in chicken DT40 cells and human TK6 cells results in hypersensitivity to Ara-C, indicating the conserved function of CTF18 in the cellular tolerance of Ara-C. Strikingly, we found that proofreading-deficient POLE1D269A/-, CTF18-/-, and POLE1D269A/-/CTF18-/- cells showed indistinguishable phenotypes, including the extent of hypersensitivity to Ara-C and decreased replication rate with Ara-C. This observed epistatic relationship between POLE1D269A/- and CTF18-/- suggests that they are interdependent in removing mis-incorporated Ara-CMP from the 3' end of primers. Mechanistically, we found that CTF18-/- cells have reduced levels of chromatin-bound Polε upon Ara-C treatment, suggesting that CTF18 contributes to the tethering of Polε on fork at the stalled end and thereby facilitating the removal of inserted Ara-C. Collectively, these data reveal the previously unappreciated role of CTF18 in Polε-exonuclease-mediated maintenance of the replication fork upon Ara-C incorporation.

The cohesin complex of yeasts: sister chromatid cohesion and beyond.

Each time a cell divides, it needs to duplicate the genome and then separate the two copies. In eukaryotes, which usually have more than one linear chromosome, this entails tethering the two newly replicated DNA molecules, a phenomenon known as sister chromatid cohesion (SCC). Cohesion ensures proper chromosome segregation to separate poles during mitosis. SCC is achieved by the presence of the cohesin complex. Besides its canonical function, cohesin is essential for chromosome organization and DNA damage repair. Surprisingly, yeast cohesin is loaded in G1 before DNA replication starts but only acquires its binding activity during DNA replication. Work in microorganisms, such as Saccharomyces cerevisiae and Schizosaccharomyces pombe has greatly contributed to the understanding of cohesin composition and functions. In the last few years, much progress has been made in elucidating the role of cohesin in chromosome organization and compaction. Here, we discuss the different functions of cohesin to ensure faithful chromosome segregation and genome stability during the mitotic cell division in yeast. We describe what is known about its composition and how DNA replication is coupled with SCC establishment. We also discuss current models for the role of cohesin in chromatin loop extrusion and delineate unanswered questions about the activity of this important, conserved complex.

PCNA Loaders and Unloaders-One Ring That Rules Them All.

During each cell duplication, the entirety of the genomic DNA in every cell must be accurately and quickly copied. Given the short time available for the chore, the requirement of many proteins, and the daunting amount of DNA present, DNA replication poses a serious challenge to the cell. A high level of coordination between polymerases and other DNA and chromatin-interacting proteins is vital to complete this task. One of the most important proteins for maintaining such coordination is PCNA. PCNA is a multitasking protein that forms a homotrimeric ring that encircles the DNA. It serves as a processivity factor for DNA polymerases and acts as a landing platform for different proteins interacting with DNA and chromatin. Therefore, PCNA is a signaling hub that influences the rate and accuracy of DNA replication, regulates DNA damage repair, controls chromatin formation during the replication, and the proper segregation of the sister chromatids. With so many essential roles, PCNA recruitment and turnover on the chromatin is of utmost importance. Three different, conserved protein complexes are in charge of loading/unloading PCNA onto DNA. Replication factor C (RFC) is the canonical complex in charge of loading PCNA during the S-phase. The Ctf18 and Elg1 (ATAD5 in mammalian) proteins form complexes similar to RFC, with particular functions in the cell's nucleus. Here we summarize our current knowledge about the roles of these important factors in yeast and mammals.

DNA Replication and Sister Chromatid Cohesion 1 (DSCC1) of the Replication Factor Complex CTF18-RFC is Critical for Colon Cancer Cell Growth.

DNA replication and sister chromatid cohesion 1 (DSCC1) combines with chromosome transmission-fidelity protein 18 (CTF18) to form a CTF18-DSCC1-CTF8 (CTF18-1-8) module, which in combination with CTF18-replication factor C (RFC) acts as a proliferating cell nuclear antigen (PCNA) loader during DNA replication-associated processes. It was found that DSCC1 was overexpressed in tumor tissues from patients with colon cancer and that the survival probability of patients with colon cancer was lower when the expression of cytosolic DSCC1 was higher in tumor regions (P=0.047). By using DSCC1- or CTF18-knockdown cell lines (HCT116-shDSCC1 or HCT116-shCTF18, respectively), it was confirmed that DSCC1-knockdown inhibits cell proliferation and invasion, but that CTF18-knockdown does not. Tumors in mice xenografted with shDSCC1 cells were significantly smaller compared with those in mice in the mock group or those xenografted with shCTF18 cells. The shDSCC1 cells were highly sensitive to γ-irradiation and other DNA replication inhibitory treatments, resulting in low cell viability. The present results suggested that DSCC1 is the most important component in the CTF18-1-8 module for CTF18-RFC and is highly relevant to the growth and metastasis of colon cancer cells, and, therefore, it may be a potential therapeutic target for colon cancer treatment.

Control of Genome Integrity by RFC Complexes; Conductors of PCNA Loading onto and Unloading from Chromatin during DNA Replication.

During cell division, genome integrity is maintained by faithful DNA replication during S phase, followed by accurate segregation in mitosis. Many DNA metabolic events linked with DNA replication are also regulated throughout the cell cycle. In eukaryotes, the DNA sliding clamp, proliferating cell nuclear antigen (PCNA), acts on chromatin as a processivity factor for DNA polymerases. Since its discovery, many other PCNA binding partners have been identified that function during DNA replication, repair, recombination, chromatin remodeling, cohesion, and proteolysis in cell-cycle progression. PCNA not only recruits the proteins involved in such events, but it also actively controls their function as chromatin assembles. Therefore, control of PCNA-loading onto chromatin is fundamental for various replication-coupled reactions. PCNA is loaded onto chromatin by PCNA-loading replication factor C (RFC) complexes. Both RFC1-RFC and Ctf18-RFC fundamentally function as PCNA loaders. On the other hand, after DNA synthesis, PCNA must be removed from chromatin by Elg1-RFC. Functional defects in RFC complexes lead to chromosomal abnormalities. In this review, we summarize the structural and functional relationships among RFC complexes, and describe how the regulation of PCNA loading/unloading by RFC complexes contributes to maintaining genome integrity.