Eiji Ohashi

Sensing and processing of DNA double-strand breaks (DSBs) are vital to genome stability. DSBs are primarily detected by the ATM checkpoint pathway, where the Mre11–Rad50–Nbs1 (MRN) complex serves as the DSB sensor. Subsequent DSB end resection activates the ATR checkpoint pathway, where replication protein A, MRN, and the Rad9–Hus1–Rad1 (9–1–1) clamp serve as the DNA structure sensors. ATR activation depends also on Topbp1, which is loaded onto DNA through multiple mechanisms. While different DNA structures elicit specific ATR-activation subpathways, the regulation and mechanisms of the ATR-activation subpathways are not fully understood. Using DNA substrates that mimic extensively resected DSBs, we show here that MRN and 9–1–1 redundantly stimulate Dna2-dependent long-range end resection and ATR activation in Xenopus egg extracts. MRN serves as the loading platform for ATM, which, in turn, stimulates Dna2- and Topbp1-loading. Nevertheless, MRN promotes Dna2-mediated end processing largely independently of ATM. 9–1–1 is dispensable for bulk Dna2 loading, and Topbp1 loading is interdependent with 9–1–1. ATR facilitates Mre11 phosphorylation and ATM dissociation. These data uncover that long-range end resection activates two redundant pathways that facilitate ATR checkpoint signaling and DNA processing in a vertebrate system.

Sensing and processing of DNA double-strand breaks (DSBs) are vital to genome stability. DSBs are primarily detected by the ATM checkpoint pathway, where the Mre11–Rad50–Nbs1 (MRN) complex serves as the DSB sensor. Subsequent DSB end resection promotes the transition from the ATM to the ATR checkpoint pathway, where replication protein A, MRN, and the Rad9–Hus1–Rad1 (9–1–1) checkpoint clamp serve as the DNA structure sensors. 9–1–1 and MRN recruit Topbp1, a critical checkpoint mediator that activates the ATR kinase. However, how multiple sensors contribute to regulating end resection and checkpoint activation remains ambiguous. Using DNA substrates that mimic extensively resected DSBs, we show here that MRN and 9–1–1 redundantly stimulate Dna2-dependent long-range end resection and ATR activation in Xenopus egg extracts. MRN serves as the loading platform for Dna2, ATM, and Topbp1. In contrast, 9–1–1 is dispensable for bulk Dna2 loading, and Topbp1 loading is interdependent with 9–1–1 in this pathway. ATR facilitates Mre11 phosphorylation and ATM dissociation. Our results delineate the molecular mechanism of and interplay between two redundant pathways that stimulate ATR checkpoint activation and long-range DSB end resection in vertebrates.

Replication initiation at specific genomic loci dictates precise duplication and inheritance of genetic information. In eukaryotic cells, ATP‐bound origin recognition complexes (ORCs) stably bind to double‐stranded (ds) DNA origins to recruit the replicative helicase onto the origin DNA. To achieve these processes, an essential region of the origin DNA must be recognized by the eukaryotic origin sensor (EOS) basic patch within the disordered domain of the largest ORC subunit, Orc1. Although ORC also binds single‐stranded (ss) DNA in an EOS‐independent manner, it is unknown whether EOS regulates ORC on ssDNA. We found that, in budding yeast, ORC multimerizes on ssDNA in vitro independently of adenine nucleotides. We also found that the ORC multimers form in an EOS‐dependent manner and stimulate the ORC ATPase activity. An analysis of genomics data supported the idea that ORC‐ssDNA binding occurs in vivo at specific genomic loci outside of replication origins. These results suggest that EOS function is differentiated by ORC‐bound ssDNA, which promotes ORC self‐assembly and ATP hydrolysis. These mechanisms could modulate ORC activity at specific genomic loci and could be conserved among eukaryotes.

Multiple regulatory roles of Rad9 C-tail in DNA damage responses, Journal of Rare Diseases Research & Treatment

The alternative proliferating-cell nuclear antigen (PCNA)-loader CTF18-RFC forms a stable complex with DNA polymerase ε (Polε). We observed that, under near-physiological conditions, CTF18-RFC alone loaded PCNA inefficiently, but loaded it efficiently when complexed with Polε. During efficient PCNA loading, CTF18-RFC and Polε assembled at a 3΄ primer–template junction cooperatively, and directed PCNA to the loading site. Site-specific photo-crosslinking of directly interacting proteins at the primer–template junction showed similar cooperative binding, in which the catalytic N-terminal portion of Polε acted as the major docking protein. In the PCNA-loading intermediate with ATPγS, binding of CTF18 to the DNA structures increased, suggesting transient access of CTF18-RFC to the primer terminus. Polε placed in DNA synthesis mode using a substrate DNA with a deoxidised 3΄ primer end did not stimulate PCNA loading, suggesting that DNA synthesis and PCNA loading are mutually exclusive at the 3΄ primer–template junction. Furthermore, PCNA and CTF18-RFC–Polε complex engaged in stable trimeric assembly on the template DNA and synthesised DNA efficiently. Thus, CTF18-RFC appears to be involved in leading-strand DNA synthesis through its interaction with Polε, and can load PCNA onto DNA when Polε is not in DNA synthesis mode to restore DNA synthesis.

Proliferating cell nuclear antigen (PCNA) and replication factor C (RFC) were identified in the late 1980s as essential factors for replication of simian virus 40 DNA in human cells, by reconstitution of the reaction in vitro. Initially, they were only thought to be involved in the elongation stage of DNA replication. Subsequent studies have demonstrated that PCNA functions as more than a replication factor, through its involvement in multiple protein-protein interactions. PCNA appears as a functional hub on replicating and replicated chromosomal DNA and has an essential role in the maintenance genome integrity in proliferating cells. Eukaryotes have multiple paralogues of sliding clamp, PCNA and its loader, RFC. The PCNA paralogues, RAD9, HUS1, and RAD1 form the heterotrimeric 9-1-1 ring that is similar to the PCNA homotrimeric ring, and the 9-1-1 clamp complex is loaded onto sites of DNA damage by its specific loader RAD17-RFC. This alternative clamp-loader system transmits DNA-damage signals in genomic DNA to the checkpoint-activation network and the DNA-repair apparatus. Another two alternative loader complexes, CTF18-RFC and ELG1-RFC, have roles that are distinguishable from the role of the canonical loader, RFC. CTF18-RFC interacts with one of the replicative DNA polymerases, Polε, and loads PCNA onto leading-strand DNA, and ELG1-RFC unloads PCNA after ligation of lagging-strand DNA. In the progression of S phase, these alternative PCNA loaders maintain appropriate amounts of PCNA on the replicating sister DNAs to ensure that specific enzymes are tethered at specific chromosomal locations.

Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as several canonical protein fractionations. Here, we present a streamlined method to construct mutant overproducers, followed by purification of mutant ORCs. Use of mammalian cells co-transfected with conveniently mutagenized plasmids bearing a His tag excludes many of the construction and fractionation steps. Transfection is highly efficient. All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex. Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells. The method is applicable to wild-type and at least three mutant ORCs, and the resultant purified complexes show expected biochemical activities. The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC.

Human Ctf18‐RFC, a PCNA loader complex, interacts with DNA polymerase ε (Polε) through a structure formed by the Ctf18, Dcc1 and Ctf8 subunits. The C‐terminal stretch of Ctf18, which is highly conserved from yeast to human, is necessary to form the Polε‐capturing structure. We found that in the budding yeast Saccharomyces cerevisiae, Ctf18, Dcc1 and Ctf8 formed the same structure through the conserved C‐terminus and interacted specifically with Polε. Thus, the specific interaction of Ctf18‐RFC with Polε is a conserved feature between these proteins. A C‐terminal deletion mutant of Ctf18 (ctf18^ΔC) exhibited the same high sensitivity to hydroxyurea as the complete deletion strain (ctf18Δ) or ATPase‐deficient mutant (ctf18^K189A), but was somewhat less sensitive to methyl methanesulfonate than either of them. These phenotypes were also observed in dcc1Δ and ctf8Δ, predicted to be deficient in the interaction with Polε. Furthermore, both plasmid loss and gross chromosomal rearrangement (GCR) rates were increased in ctf18^ΔC cells to the same extent as in ctf18Δ cells. These results indicate that the Ctf18‐RFC/Polε interaction plays a crucial role in maintaining genome stability in budding yeast, probably through recruitment of this PCNA loader to the replication fork.

In eukaryotes, the origin recognition complex (ORC) heterohexamer preferentially binds replication origins to trigger initiation of DNA replication. Crystallographic studies using eubacterial and archaeal ORC orthologs suggested that eukaryotic ORC may bind to origin DNA via putative winged-helix DNA-binding domains and AAA+ ATPase domains. However, the mechanisms how eukaryotic ORC recognizes origin DNA remain elusive. Here, we show in budding yeast that Lys-362 and Arg-367 residues of the largest subunit (Orc1), both outside the aforementioned domains, are crucial for specific binding of ORC to origin DNA. These basic residues, which reside in a putative disordered domain, were dispensable for interaction with ATP and non-specific DNA sequences, suggesting a specific role in recognition. Consistent with this, both residues were required for origin binding of Orc1 in vivo. A truncated Orc1 polypeptide containing these residues solely recognizes ARS sequence with low affinity and Arg-367 residue stimulates sequence specific binding mode of the polypeptide. Lys-362 and Arg-367 residues of Orc1 are highly conserved among eukaryotic ORCs, but not in eubacterial and archaeal orthologs, suggesting a eukaryote-specific mechanism underlying recognition of replication origins by ORC.