Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1.

Post-replicative correction of replication errors by the mismatch repair (MMR) system is critical for suppression of mutations. Although the MMR system may need to handle nucleosomes at the site of chromatin replication, how MMR occurs in the chromatin environment remains unclear. Here, we show that nucleosomes are excluded from a >1-kb region surrounding a mismatched base pair in Xenopus egg extracts. The exclusion was dependent on the Msh2-Msh6 mismatch recognition complex but not the Mlh1-containing MutL homologs and counteracts both the HIRA- and CAF-1 (chromatin assembly factor 1)-mediated chromatin assembly pathways. We further found that the Smarcad1 chromatin remodeling ATPase is recruited to mismatch-carrying DNA in an Msh2-dependent but Mlh1-independent manner to assist nucleosome exclusion and that Smarcad1 facilitates the repair of mismatches when nucleosomes are preassembled on DNA. In budding yeast, deletion of FUN30, the homolog of Smarcad1, showed a synergistic increase of spontaneous mutations in combination with MSH6 or MSH3 deletion but no significant increase with MSH2 deletion. Genetic analyses also suggested that the function of Fun30 in MMR is to counteract CAF-1. Our study uncovers that the eukaryotic MMR system has an ability to exclude local nucleosomes and identifies Smarcad1/Fun30 as an accessory factor for the MMR reaction.

Rituximab in combination with cyclosporine and steroid pulse therapy for childhood-onset multidrug-resistant nephrotic syndrome: a multicenter single-arm clinical trial (JSKDC11 trial).

BACKGROUND: Only 80% of children with idiopathic nephrotic syndrome respond well to glucocorticoid therapy. Multidrug-resistant nephrotic syndrome (MRNS) is associated with a poor kidney prognosis. Several retrospective studies have identified rituximab as an effective treatment for MRNS; however, prospective studies are required to assess its efficacy and safety. METHODS: We conducted a multicenter, non-blinded, single-arm trial to investigate the efficacy and safety of rituximab in patients with childhood-onset MRNS who were resistant to cyclosporine and more than three courses of steroid pulse therapy. The enrolled patients received four 375 mg/m2 doses of rituximab in combination with baseline cyclosporine and steroid pulse therapy. The primary endpoint was a > 50% reduction in the urinary protein/creatinine ratio from baseline on day 169. Complete and partial remissions were also evaluated. RESULTS: Six patients with childhood-onset MRNS were enrolled. All patients were negative for pathogenic variants of podocyte-related genes. On day 169, five patients (83.3%) showed a > 50% reduction in the urinary protein/creatinine ratio, two patients showed partial remission, and two patients showed complete remission. No deaths occurred and severe adverse events occurred in two patients (infection in one patient and acute kidney injury in one patient). Three patients needed treatment for moderate-to-severe infection. CONCLUSIONS: The study treatment effectively reduced the urinary protein/creatinine ratio in patients with childhood-onset MRNS. The adverse events in this study were within the expected range; however, attention should be paid to the occurrence of infections.

Systemic inflammation caused by an intracranial mesenchymal tumor with a EWSR1::CREM fusion presenting associated with IL-6/STAT3 signaling.

Pediatric neoplastic diseases account for about 10% of cases of fever of unknown origin (FUO), and most neoplastic disease cases are leukemia, lymphoma, and neuroblastoma. Brain tumors are rarely reported as the cause of FUO, although craniopharyngioma, metastatic brain tumor, and Castleman's disease have been reported. We report a case of intracranial mesenchymal tumor (IMT) with a FET:CREB fusion gene, which had inflammatory phenotype without neurological signs. A 10-year-old girl was admitted with a 2-month history of intermittent fever and headache, whereas her past history as well as her family history lacked special events. Sepsis work-up showed no pathological organism, and empirical antibiotic therapy was not effective. Bone marrow examination showed a negative result. Cerebrospinal fluid examination showed elevated protein as well as cell counts, and head magnaetic resonance imaging showed a hypervascular mass lesion with contrast enhancement in the left cerebellar hemisphere. The patient underwent tumor excision, which made the intermittent fever disappear. Pathological examinations resembled those of classic angiomatoid fibrous histiocytoma (AFH), but the morphological features were distinct from the AFH myxoid variant; then we performed break-apart fluorescence in situ hybridization and confirmed the tumor harbored the rare EWSR1::CREM fusion gene (Ewing sarcoma breakpoint region 1 gene (EWSR1) and cAMP response element binding (CREB) family gene). Consequently, we diagnosed the condition as IMT with EWSR1::CREM fusion. Elevated serum concentration of interleukin 6 (IL-6) was normalized after tumor resection, which suggested the fever could be caused by tumor-derived IL-6. This is the first case of IMT with EWSR1::CREM fusion that showed paraneoplastic symptoms associated with the IL-6/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Although brain tumors are rarely diagnosed as a responsible disease for FUO, they should be considered as a cause of unknown fever even in the absence of abnormal neurological findings.

Mycophenolate Mofetil after Rituximab for Childhood-Onset Complicated Frequently-Relapsing or Steroid-Dependent Nephrotic Syndrome.

BACKGROUND: Rituximab is the standard therapy for childhood-onset complicated frequently relapsing or steroid-dependent nephrotic syndrome (FRNS/SDNS). However, most patients redevelop FRNS/SDNS after peripheral B cell recovery. METHODS: We conducted a multicenter, randomized, double-blind, placebo-controlled trial to examine whether mycophenolate mofetil (MMF) administration after rituximab can prevent treatment failure (FRNS, SDNS, steroid resistance, or use of immunosuppressive agents or rituximab). In total, 39 patients (per group) were treated with rituximab, followed by either MMF or placebo until day 505 (treatment period). The primary outcome was time to treatment failure (TTF) throughout the treatment and follow-up periods (until day 505 for the last enrolled patient). RESULTS: TTFs were clinically but not statistically significantly longer among patients given MMF after rituximab than among patients receiving rituximab monotherapy (median, 784.0 versus 472.5 days, hazard ratio [HR], 0.59; 95% confidence interval [95% CI], 0.34 to 1.05, log-rank test: P=0.07). Because most patients in the MMF group presented with treatment failure after MMF discontinuation, we performed a post-hoc analysis limited to the treatment period and found that MMF after rituximab prolonged the TTF and decreased the risk of treatment failure by 80% (HR, 0.20; 95% CI, 0.08 to 0.50). Moreover, MMF after rituximab reduced the relapse rate and daily steroid dose during the treatment period by 74% and 57%, respectively. The frequency and severity of adverse events were similar in both groups. CONCLUSIONS: Administration of MMF after rituximab may sufficiently prevent the development of treatment failure and is well tolerated, although the relapse-preventing effect disappears after MMF discontinuation.

Advanced Solid-State NMR Analysis of Two Crystal Forms of Ranitidine Hydrochloride: Detection of 1H-14N Intra-/Intermolecular Correlations.

Understanding the characteristics of crystal polymorphism of active pharmaceutical ingredients and analyzing them with high sensitivity is important for quality of drug products, appropriate characterization strategies, and appropriate screening and selection processes. However, there are few methods to measure intra- and intermolecular correlations in crystals other than X-ray crystallography, for which it is sometimes difficult to obtain suitable single crystals. Recently, solid-state NMR has been recognized as a straightforward method for measuring molecular correlations. In this study, we selected ranitidine hydrochloride, which is known to exist in two forms, 1 and 2, as the model drug and investigated each form using solid-state NMR. In conducting the analysis, rotating the sample tube, which had a 1-mm inner diameter, increased the solid-state NMR resolution at 70 kHz. The 1H-14N dipolar-based heteronuclear multiple quantum coherence (D-HMQC) analysis revealed the intermolecular correlation of Form 1 between the N atom of the nitro group and a proton of the furan moiety, which were closer than those of the intramolecular correlation reported using single X-ray crystal analysis. Thus, 1H-14N D-HMQC analysis could be useful for characterizing intermolecular interaction in ranitidine hydrochloride crystals. In addition, we reassigned the 13C solid-state NMR signals of ranitidine hydrochloride according to the liquid-state and multiple solid-state NMR experiments.

Immunoglobulin G deposition on proximal tubules and the tubular basement membrane in acute tubular injury complicated with focal segmental glomerulosclerosis (FSGS): A possible prediction tool for subclinical FSGS.

Immunofluorescent deposition of immunoglobulin G (IgG) in the tubular basement membrane (TBM) has been evaluated in the diagnosis of various diseases; however, few studies have investigated the immunofluorescence of acute tubular injury (ATI). Herein, we attempted to clarify IgG expression in the proximal tubular epithelium and TBM in ATI due to various causes. Patients with ATI with nephrotic-range proteinuria, including focal segmental glomerulosclerosis (FSGS, n = 18) and minimal change nephrotic syndrome (MCNS, n = 8), ATI with ischemia (n = 6), and drug-induced ATI (n = 7), were enrolled. ATI was evaluated by light microscopy. CD15 and IgG double staining and IgG subclass staining were performed to evaluate immunoglobulin deposition in the proximal tubular epithelium and TBM. IgG deposition was identified in the proximal tubules only in the FSGS group. Furthermore, IgG deposition in the TBM was observed in the FSGS group showing severe ATI. IgG3 was predominantly deposited by the IgG subclass study. Our results indicate that IgG deposition in the proximal tubular epithelium and TBM suggests the leaking of IgG from the glomerular filtration barrier and its reabsorption by proximal tubules, which may predict disruption of the glomerular size barrier, including subclinical FSGS. FSGS with ATI should be included as a differential diagnosis when IgG deposition in TBM is observed.

Flavonoids from Sedum japonicum subsp. oryzifolium (Crassulaceae).

Twenty-two flavonoids were isolated from the leaves and stems of Sedum japonicum subsp. oryzifolium (Crassulaceae). Of these compounds, five flavonoids were reported in nature for the first time, and identified as herbacetin 3-O-xyloside-8-O-glucoside, herbacetin 3-O-glucoside-8-O-(2'''-acetylxyloside), gossypetin 3-O-glucoside-8-O-arabinoside, gossypetin 3-O-glucoside-8-O-(2'''-acetylxyloside) and hibiscetin 3-O-glucoside-8-O-arabinoside via UV, HR-MS, LC-MS, acid hydrolysis and NMR. Other seventeen known flavonoids were identified as herbacetin 3-O-glucoside-8-O-arabinoside, herbacetin 3-O-glucoside-8-O-xyloside, gossypetin 3-O-glucoside-8-O-xyloside, quercetin, quercetin 3-O-glucoside, quercetin 3-O-xylosyl-(1→2)-rhamnoside-7-O-rhamnoside, quercetin 3-O-rhamnoside-7-O-glucoside, kaempferol, kaempferol 3-O-glucoside, kaempferol 7-O-rhamnoside, kaempferol 3,7-di-O-rhamnoside, kaempferol 3-O-glucoside-7-O-rhamnoside, kaempferol 3-O-glucosyl-(1→2)-rhamnoside-7-O-rhamnoside, kaempferol 3-O-xylosyl-(1→2)-rhamnoside, kaempferol 3-O-xylosyl-(1→2)-rhamnoside-7-O-rhamnoside, myricetin 3-O-glucoside and cyanidin 3-O-glucoside. Some flavonol 3,8-di-O-glycosides were found in Sedum japonicum subsp. oryzifolium as major flavonoids in this survey. They were presumed to be the diagnostic flavonoids in the species. Flavonoids were reported from S. japonicum for the first time.

Isolation of New Colibactin Metabolites from Wild-Type Escherichia coli and In Situ Trapping of a Mature Colibactin Derivative.

Colibactin is a polyketide-nonribosomal peptide hybrid secondary metabolite that can form interstrand cross-links in double-stranded DNA. Colibactin-producing Escherichia coli has also been linked to colorectal oncogenesis. Thus, there is a strong interest in understanding the role colibactin may play in oncogenesis. Here, using the high-colibactin-producing wild-type E. coli strain we isolated from a clinical sample with the activity-based fluorescent probe we developed earlier, we were able to identify colibactin 770, which was recently identified and proposed as the complete form of colibactin, along with colibactin 788, 406, 416, 420, and 430 derived from colibactin 770 through structural rearrangements and solvolysis. Furthermore, we were able to trap the degrading mature colibactin species by converting the diketone moiety into quinoxaline in situ in the crude culture extract to form colibactin 860 at milligram scale. This allowed us to determine the stereochemically complex structure of the rearranged form of an intact colibactin, colibactin 788, in detail. Furthermore, our study suggested that we were capturing only a few percent of the actual colibactin produced by the microbe, providing a crude quantitative insight into the inherent instability of this compound. Through the structural assignment of colibactins and their degradative products by the combination of LC-HRMS and NMR spectroscopies, we were able to elucidate further the fate of inherently unstable colibactin, which could help acquire a more complete picture of colibactin metabolism and identify key DNA adducts and biomarkers for diagnosing colorectal cancer.

Quantitative Analysis of Ent-Kaurane Diterpenoids in Isodon Herb (Enmei-So) by HPLC-UV.

The terrestrial plants, Isodon japonicus (Burm. f.) H. Hara and Isodon trichocarpus (Maxim.) Kudô (Labiatae), are native to Japan. Different parts of these plants have been used as a traditional bitter stomachic, under the name Isodon herb (Enmei-so). Ent-kaurane diterpenoids are the major constituents of Isodon herb that contribute to the herb's medicinal properties. However, large variability with respect to the composition of these diterpenoids limits the suitability of Isodon herb as a pharmaceutical ingredient. Thus, an investigation of the factors that affect its chemical composition is required. In this study, the DNA-barcoding method, using internal transcribed spacer sequences of nuclear ribosomal DNA, was applied to cultivated and commercial samples of Isodon herb. Further, each such sample was separated into leaves, stems, and flowers and analyzed for diterpenoid content by HPLC. Moreover, the diterpenoid content in coarsely cut and powdered samples was evaluated. Results confirmed that the source species of these samples was I. japonicus or I. trichocarpus. The three major diterpenoids in Isodon herb were enmein, oridonin, and ponicidin. The diterpenoid content was affected by milling process. Moreover, the diterpenoid content was greatly affected by the ratio between leaves and stems in each sample. Thus, to accurately quantify the diterpenoids in Isodon herb, the use specific conditions such as drying using mild temperature conditions and avoiding milling of the samples might be necessary. This may help in regulating variations in the herb's composition, in turn, providing better quality and a safe herbal product for pharmaceutical use.

Recombinant yellow protein of the takeout family and albino-related takeout protein specifically bind to lutein in the desert locust.

Yellow protein of the takeout family (YPT) and albino-related takeout protein (ALTO) are involved in body-color polyphenism in Schistocerca gregaria. YPT has been proposed to bind to ß-carotene, whereas the physiological role of ALTO is unclear. Structurally, takeout proteins contain a long continuous tunnel to bind specific ligands. However, the specific ligands of YPT and ALTO have not been fully elucidated. Here, we isolated the full coding cDNAs of these proteins and successfully produced recombinant YPT and ALTO using an Escherichia coli expression system. Absorption spectral analyses of YPT with and without carotenoids revealed that this protein bound to lutein. In contrast, obvious binding of YPT to ß-carotene and astaxanthin was not detected. Similar results were obtained for ALTO. The presence of juvenile hormone only weakly affected the protein/carotenoid interactions. These results suggested that YPT and ALTO specifically bound to lutein in a juvenile hormone-independent manner.

An efficient method for the isolation of interaction-null/impaired mutants using the yeast two-hybrid technique.

Protein-protein interactions are one of the most basic and critical processes underlying biological functions. Thus, identification of the interacting proteins of a protein of interest and further elucidation of the roles of the interactions is critical for understanding the related biological processes. The yeast two-hybrid (Y2H) method is a popular approach for identifying protein-protein interactions. Once interacting proteins are identified, a comparison of the phenotypes of mutants lacking the specific protein-protein interaction with those of the wild-type strain is a powerful tool for uncovering the former interactions' biological significance. However, isolation of such interaction-defective mutants is often laborious. Here, I describe a novel and efficient approach for isolating such mutants that uses the Y2H technique with a modified Y2H vector, and provide an example of how this approach can be used to screen interaction-null/impaired mutants. Because the strategy is simple and the modification of a pre-existing Y2H vector is sufficient for the screening purpose, the same strategy can be applied to any existing two-hybrid system.

Two separate pathways regulate protein stability of ATM/ATR-related protein kinases Mec1 and Tel1 in budding yeast.

Checkpoint signaling requires two conserved phosphatidylinositol 3-kinase-related protein kinases (PIKKs): ATM and ATR. In budding yeast, Tel1 and Mec1 correspond to ATM and ATR, respectively. The Tel2-Tti1-Tti2 (TTT) complex connects to the Rvb1-Rvb2-Tah1-Pih1 (R2TP) complex for the protein stability of PIKKs; however, TTT-R2TP interaction only partially mediates ATM and ATR protein stabilization. How TTT controls protein stability of ATM and ATR remains to be precisely determined. Here we show that Asa1, like Tel2, plays a major role in stabilization of newly synthesized Mec1 and Tel1 proteins whereas Pih1 contributes to Mec1 and Tel1 stability at high temperatures. Although Asa1 and Pih1 both interact with Tel2, no Asa1-Pih1 interaction is detected. Pih1 is distributed in both the cytoplasm and nucleus wheres Asa1 localizes largely in the cytoplasm. Asa1 and Pih1 are required for proper DNA damage checkpoint signaling. Our findings provide a model in which two different Tel2 pathways promote protein stabilization of Mec1 and Tel1 in budding yeast.

Pre-initiation complex assembly functions as a molecular switch that splits the Mcm2-7 double hexamer.

Initiation of chromosomal DNA replication in eukaryotes involves two steps: licensing and firing. In licensing, a core component of the replicative helicase, the Mcm2-7 complex, is loaded onto replication origins as an inactive double hexamer, which is activated in the firing step by firing factors. A reaction intermediate called the pre-initiation complex (pre-IC) has been proposed to assemble transiently during firing, but the existence of the pre-IC has not yet been confirmed. Here, we show, by systematic chromatin immunoprecipitation, that a distinct intermediate that fits the definition of the pre-IC assembles during firing in the budding yeast Saccharomyces cerevisiae Pre-IC assembly is observed in the absence of Mcm10, one of the firing factors, and is mutually dependent on all the firing factors whose association to replication origins is triggered by cyclin-dependent kinase. In the pre-IC, the Mcm2-7 double hexamer is separated into single hexamers, as in the active helicase. Our data indicate that pre-IC assembly functions as an all-or-nothing molecular switch that splits the Mcm2-7 double hexamer.

Yellowing and YPT gene expression in the desert locust, Schistocerca gregaria: Effects of developmental stages and fasting.

The yellow protein of the takeout family (YPT) controls the development of yellow body color in the desert locust. This study focused on two aspects related to YPT in the locust. We first examined the expression pattern of YPT during nymphal stages because yellowing was not obvious during the early instars. YPT expression levels were extremely low in the second and third instars compared with the last two nymphal instars. Warm rearing temperature and juvenile hormone (JH) injection, which stimulated YPT expression in the late instars, had little effect in the second instar, suggesting that YPT expression during the early instars was suppressed and could not be stimulated by either of these factors. We also investigated delayed yellowing in fasting male adults, under the hypothesis that fasting decreased the JH titers and delayed the onset of YPT expression. Yellowing was delayed in fasting adults compared with well-fed adults and YPT expression was stimulated by JH injections at Day 15. However, we failed to obtain evidence that fasting significantly influenced the expression levels of YPT and the JH early-inducible gene Krüppel homolog 1 at Days 15 and 20 post-adult emergence. Results suggest that a YPT-independent mechanism possibly induces delayed yellowing in fasting males.

RNAi-mediated knockdown of SPOOK reduces ecdysteroid titers and causes precocious metamorphosis in the desert locust Schistocerca gregaria.

The Halloween gene SPOOK (SPO) is involved in the production of the active metabolite of ecdysteroid, 20-hydroxyecdysone (20E), in insects. A previous study showed that RNAi-mediated knockdown of SPO in Schistocerca gregaria last instar nymphs markedly reduced the hemolymph 20E titer, but did not affect metamorphosis. In the present study, the effects of SPO interference on development were re-examined in this locust. Injections of SPO double-stranded RNA (dsSPO) into nymphs at mid and late instars significantly delayed nymphal development and interfered with molting. The 20E levels of dsSPO-treated nymphs were generally low, with a delayed, small peak, suggesting that disturbance of the 20E levels caused the above developmental abnormalities. A small proportion of the dsSPO-injected nymphs metamorphosed precociously, producing adults and adultoids. Precocious adults were characterized by small body size, short wings with abbreviated venation, and normal reproductive activity. Fourth instar nymphs that precociously metamorphosed at the following instar exhibited temporal expression patterns of ecdysone-induced protein 93F and the juvenile hormone (JH) early-inducible gene Krüppel homolog 1 similar to those observed at the last instar in normal nymphs. Adultoids displayed mating behavior and adultoid females developed eggs, but never laid eggs. JH injection around the expected time of the 20E peak in the dsSPO-injected nymphs completely inhibited the appearance of adultoids, suggesting that appearance of adultoids might be due to a reduced titer of JH rather than of 20E. These results suggest that SPO plays an important role in controlling morphogenesis, metamorphosis, and reproduction in S. gregaria.

Conserved interaction of Ctf18-RFC with DNA polymerase ε is critical for maintenance of genome stability in Saccharomyces cerevisiae.

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.

Japanese Medical Education Reforms during the Allied Forces Occupancy; Roles Played by the Public Health and Welfare Section of the Supreme Commander for the Allied Powers.

The health and welfare of the Japanese people were of a lower standard compared to other developed countries at the end of the World War II in 1945. Crawford F. Sams, Chief, the Public Health and Welfare Section of the Supreme Commander for the Allied Powers thought that medical care in a wartom country could be improved not by building new hospitals and providing more medical equipment, but through professional education and training. He founded the Council on Medical Education to reform the Japanese medical education. The Council shaped Japanese medical education by establishing the standards for medical school education and initiating internship and a national medical licensure examination. In the early 1950s, the Unitarian Service Committee Medical Mission was invited to teach to medical school professors and students American medicine. This medical mission was also a contribution of the Public Health and Welfare Section to Japanese medical education. This article explores how Public Health and Welfare Section played vital roles in transforming Japanese medical education and postgraduate training during the occupation.

iAID: an improved auxin-inducible degron system for the construction of a 'tight' conditional mutant in the budding yeast Saccharomyces cerevisiae.

Isolation of a 'tight' conditional mutant of a gene of interest is an effective way of studying the functions of essential genes. Strategies that use ubiquitin-mediated protein degradation to eliminate the product of a gene of interest, such as heat-inducible degron (td) and auxin-inducible degron (AID), are powerful methods for constructing conditional mutants. However, these methods do not work with some genes. Here, we describe an improved AID system (iAID) for isolating tight conditional mutants in the budding yeast Saccharomyces cerevisiae. In this method, transcriptional repression by the 'Tet-OFF' promoter is combined with proteolytic elimination of the target protein by the AID system. To provide examples, we describe the construction of tight mutants of the replication factors Dpb11 and Mcm10, dpb11-iAID, and mcm10-iAID. Because Dpb11 and Mcm10 are required for the initiation of DNA replication, their tight mutants are unable to enter S phase. This is the case for dpb11-iAID and mcm10-iAID cells after the addition of tetracycline and auxin. Both the 'Tet-OFF' promoter and the AID system have been shown to work in model eukaryotes other than budding yeast. Therefore, the iAID system is not only useful in budding yeast, but also can be applied to other model systems to isolate tight conditional mutants.

Multiple regulatory mechanisms to inhibit untimely initiation of DNA replication are important for stable genome maintenance.

Genomic instability is a hallmark of human cancer cells. To prevent genomic instability, chromosomal DNA is faithfully duplicated in every cell division cycle, and eukaryotic cells have complex regulatory mechanisms to achieve this goal. Here, we show that untimely activation of replication origins during the G1 phase is genotoxic and induces genomic instability in the budding yeast Saccharomyces cerevisiae. Our data indicate that cells preserve a low level of the initiation factor Sld2 to prevent untimely initiation during the normal cell cycle in addition to controlling the phosphorylation of Sld2 and Sld3 by cyclin-dependent kinase. Although untimely activation of origin is inhibited on multiple levels, we show that deregulation of a single pathway can cause genomic instability, such as gross chromosome rearrangements (GCRs). Furthermore, simultaneous deregulation of multiple pathways causes an even more severe phenotype. These findings highlight the importance of having multiple inhibitory mechanisms to prevent the untimely initiation of chromosome replication to preserve stable genome maintenance over generations in eukaryotes.