Chl12 (Ctf18) Forms a Novel Replication Factor C-Related Complex and Functions Redundantly with Rad24 in the DNA Replication Checkpoint Pathway

Abstract

RAD24 has been identified as a gene essential for the DNA damage checkpoint in budding yeast. Rad24 is structurally related to subunits of the replication factor C (RFC) complex, and forms an RFC-related complex with Rfc2, Rfc3, Rfc4, and Rfc5. The rad24⌬ mutation enhances the defect of rfc5-1 in the DNA replication block checkpoint, implicating RAD24 in this checkpoint. CHL12 (also called CTF18) encodes a protein that is structurally related to the Rad24 and RFC proteins. We show here that although neither chl12⌬ nor rad24⌬ single mutants are defective, chl12⌬ rad24⌬ double mutants become defective in the replication block checkpoint. We also show that Chl12 interacts physically with Rfc2, Rfc3, Rfc4, and Rfc5 and forms an RFC-related complex which is distinct from the RFC and RAD24 complexes. Our results suggest that Chl12 forms a novel RFC-related complex and functions redundantly with Rad24 in the DNA replication block checkpoint.Eukaryotic cells employ a set of surveillance mechanisms to coordinate the onset of one event and the completion of the preceding event during the cell cycle. The mechanisms that ensure the proper ordering of cell cycle events have been termed checkpoint controls in eukaryotes (11). When DNA is damaged or DNA replication is blocked, the activation of checkpoint pathways arrests the cell cycle and induces the transcription of genes that facilitate DNA repair and/or replication (5, 33).Checkpoint pathways are an evolutionarily conserved feature of eukaryotic cells. This feature is typified in the ATM and ATR family genes which encode phosphatidylinositol 3-kinaserelated proteins possessing protein kinase activity (33). In the budding yeast Saccharomyces cerevisiae, MEC1 encodes an ATR-related protein and plays a critical role in checkpoint controls (14,17,32). Mec1 physically interacts with Pie1 (also called Lcd1 or Ddc2), a protein that exhibits limited homology to the fission yeast Rad26 protein (17,19,32). Likewise, in fission yeast the ATR family protein Rad3 forms a complex with Rad26 (4). DNA damage responses have been well characterized in budding yeast and consist of the G 1 -, S-, and G 2 /M-phase damage checkpoints (14). Both Mec1 and Pie1 are essential for all three DNA damage checkpoints, as well as the DNA replication block checkpoint.In addition to MEC1 and PIE1, a number of genes that control the checkpoints in budding yeast have been identified. These include DDC1, MEC3, RAD9, RAD17, RAD24, and RAD53 (5,14,33). RAD53 encodes a protein kinase and functions downstream of MEC1 in the checkpoint pathway. Like Mec1, Rad53 plays an essential role in both the replication block and DNA damage checkpoints. Following DNA damage and replication block, the Rad53 protein is hyperphosphorylated and activated by a mechanism dependent on Mec1 (20, 26). Thus, Mec1 and Rad53 constitute a central checkpoint pathway in budding yeast. RAD9, RAD17, MEC3, DDC1, and RAD24 are also required for DNA damage checkpoints. Rad9 is hyperphosphorylated following DNA damage, and the phosphorylated Ra...

A Second Proliferating Cell Nuclear Antigen Loader Complex, Ctf18-Replication Factor C, Stimulates DNA Polymerase η Activity

“…Previous work demonstrated that stalling of pol ␦ and/or ⑀ leads to checkpoint signaling through the redundant functions of Rad17-RFC and Ctf18-RFC (56). A genome-wide DNA integrity network study also demonstrated the importance of Ctf18-RFC in the DNA damage response pathway (57).…”

Genetic Analysis of Saccharomyces cerevisiae H2A Serine 129 Mutant Suggests a Functional Relationship Between H2A and the Sister-Chromatid Cohesion Partners Csm3–Tof1 for the Repair of Topoisomerase I-Induced DNA Damage

“…One function of Ctf18-RFC is to initiate switching to DNA polymerase-s during replication (Mayer et al 2004). Moreover, like the Csm3-Tof1 complex, Ctf8-RFC is required for proper sister-chromatid cohesion (Hanna et al 2001;Mayer et al 2001;Naiki et al 2001). As Csm3 and Tof1 are lethal with Ctf8, a partner of Dcc1, we hypothesized that an alteration of H2A-S129, in combination with DCC1 deletion, would show synthetic interaction and/ or would confer additional sensitivity to CPT.…”

Telomerase Is Essential to Alleviate Pif1-Induced Replication Stress at Telomeres

“…Alternatively, Sgs1 may be involved in repairing damage caused by PIF1 overexpression. Ctf18, Dcc1, and Ctf8, along with Rfc2-5, form an alternative RFC complex that has been linked to the DNA damage response and sister chromatid cohesion (Mayer et al 2001;Naiki et al 2001). Asf1 is a histone chaperone that functions with Rtt109, a histone acetyltransferase, to acetylate lysine K56 on histone H3 (Collins et al 2007;Driscoll et al 2007), which is critical for chromatin reassembly following DSB repair (Chen et al 2008).…”