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MCM2 and MCM3 are two genetically interacting and structurally related proteins essential for growth inSaccharomyces cerevisiae. Mutants defective in these proteins affect the stability of minichromosomes in general, but the severity of the defect is dependent on the autonomously replicating sequence (ARS) that drives the replication of that plasmid. In this paper we show by two-dimensional gel electrophoresis that the initiation of DNA synthesis at chromosomal replication origins is also reduced in frequency in these mutants. We show further that the nuclear and subnuclear localizations of the MCM2 and MCM3 proteins are temporally regulated with respect to the cell cycle. These proteins enter the nucleus at the end of mitosis, persist there throughout G~ phase, and disappear from it at the beginning of S phase. Once inside the nucleus, a fraction of the MCM2 and MCM3 proteins becomes tightly associated with DNA. The association of MCM2 and MCM3 with chromatin presumably leads to the initiation of DNA synthesis, and their subsequent disappearance from the nucleus presumably prevents reinitiation of DNA synthesis at replication origins. This temporally and spatially restricted localization of MCM2 and MCM3 in the nucleus may serve to ensure that DNA replication occurs once and only once per cell cycle.[Key Words: MCM2; MCM3; S. cerevisiae; nuclear localization; initiation of DNA synthesis; DNA replication; licensing factors] Received July 13, 1993; revised version accepted August 24, 1993.Regulation of DNA replication in eukaryotes is dovetailed with stages of the cell cycle. In each cell cycle, (1) replication occurs only during S phase, (2) chromosomal re-replication is prevented, (3) mitosis is delayed until replication is completed, and (4) the re-replication block is removed before the next S phase. The coupling between completion of DNA replication and mitosis is relatively well understood. Replicating DNA sends out a negative signal that suppresses the activation of MPF, the mitosis (or maturation) promoting factor (Dasso and Newport 1990; Smythe and Newport 1992). In contrast, little is known about other mechanisms that coordinate DNA replication with the cell cycle.Using Xenopus egg extracts, Blow and Laskey (1988) found that to get a second round of replication, nuclei either have to go through mitosis, during which the nuclear envelope breaks down and reassembles, or be permeabilized with nonionic detergents. This observation suggests the existence of an essential replication factor, called licensing factor, which is destroyed immediately after replication initiation and which cannot gain access into the nucleus unless the nuclear envelope is perturbed. In this way, cells can effectively block re-replication of their genome and then erase the block by going through mitosis. The concept of a licensing factor can easily explain the results of earlier cell fusion studies that G1 nuclei, but not G2 nuclei, can initiate DNA replication when fused with S cells (Rao and Johnson 1970). However, this hypothesis...

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Using cell-free extracts made from Xenopus eggs, we show that cdk2-cyclin E and A kinases play an important role in negatively regulating DNA replication. Specifically, we demonstrate that the cdk2 kinase concentration surrounding chromatin in extracts increases 200-fold once the chromatin is assembled into nuclei. Further, we find that if the cdk2–cyclin E or A concentration in egg cytosol is increased 16-fold before the addition of sperm chromatin, the chromatin fails to initiate DNA replication once assembled into nuclei. This demonstrates that cdk2–cyclin E or A can negatively regulate DNA replication. With respect to how this negative regulation occurs, we show that high levels of cdk2–cyclin E do not block the association of the protein complex ORC with sperm chromatin but do prevent association of MCM3, a protein essential for replication. Importantly, we find that MCM3 that is prebound to chromatin does not dissociate when cdk2– cyclin E levels are increased. Taken together our results strongly suggest that during the embryonic cell cycle, the low concentrations of cdk2–cyclin E present in the cytosol after mitosis and before nuclear formation allow proteins essential for potentiating DNA replication to bind to chromatin, and that the high concentration of cdk2–cyclin E within nuclei prevents MCM from reassociating with chromatin after replication. This situation could serve, in part, to limit DNA replication to a single round per cell cycle.

Background. Non-small cell lung cancer patients with epidermal growth factor receptor (EGFR) mutations have mixed responses to tyrosine kinase inhibitors (TKIs). Intertumor heterogeneity in EGFR mutations is one potential explanation for this phenomenon.Methods. We performed direct sequencing to identify EGFR mutations in 180 pairs of lung adenocarcinoma samples (from 3,071 patients). The high-resolution melting method was used in discordant cases to confirm EGFR mutation status. Matching samples were divided into four groups: primary lesions detected at different times, primary tumors with matched metastatic lymph nodes, multiple pulmonary nodules, and primary tumors with matched distant metastases. Multivariate analyses were performed to evaluate correlations between heterogeneity and patient characteristics.Results. In the study population, the discordance rate

BackgroundAppropriate patient selection is needed for targeted therapies that are efficacious only in patients with specific genetic alterations. We aimed to define subgroups of patients with candidate driver genes in patients with non-small cell lung cancer.MethodsPatients with primary lung cancer who underwent clinical genetic tests at Guangdong General Hospital were enrolled. Driver genes were detected by sequencing, high-resolution melt analysis, qPCR, or multiple PCR and RACE methods.Results524 patients were enrolled in this study, and the differences in driver gene alterations among subgroups were analyzed based on histology and smoking status. In a subgroup of non-smokers with adenocarcinoma, EGFR was the most frequently altered gene, with a mutation rate of 49.8%, followed by EML4-ALK (9.3%), PTEN (9.1%), PIK3CA (5.2%), c-Met (4.8%), KRAS (4.5%), STK11 (2.7%), and BRAF (1.9%). The three most frequently altered genes in a subgroup of smokers with adenocarcinoma were EGFR (22.0%), STK11 (19.0%), and KRAS (12.0%). We only found EGFR (8.0%), c-Met (2.8%), and PIK3CA (2.6%) alterations in the non-smoker with squamous cell carcinoma (SCC) subgroup. PTEN (16.1%), STK11 (8.3%), and PIK3CA (7.2%) were the three most frequently enriched genes in smokers with SCC. DDR2 and FGFR2 only presented in smokers with SCC (4.4% and 2.2%, respectively). Among these four subgroups, the differences in EGFR, KRAS, and PTEN mutations were statistically significant.ConclusionThe distinct features of driver gene alterations in different subgroups based on histology and smoking status were helpful in defining patients for future clinical trials that target these genes. This study also suggests that we may consider patients with infrequent alterations of driver genes as having rare or orphan diseases that should be managed with special molecularly targeted therapies.

The initiation of DNA replication involves a minimum of four factors: a specific DNA sequence (origin), an initiator protein which binds to the origin, a helicase that unwinds the origin and a protein that binds single-stranded DNA that stabilizes the unwound origin. In eukaryotic cells, the origin recognition complex (ORC) is the initiator protein and replication protein A (RPA; ref. 3) is the single-stranded DNA-binding protein. However, the helicase has not been identified and the nature of origins remains elusive, except in the case of Saccharomyces cerevisiae. A unique feature of eukaryotic DNA replication is that it occurs at a few-hundred discrete foci. It has thus been proposed that a real origin must contain a specific DNA sequence and must be attached to replication foci. Using Xenopus laevis egg extracts, we have identified and purified a 170-kD protein, focus-forming activity 1 (FFA-1), which is required for the formation of replication foci. Here we report that FFA-1 has DNA-helicase activity. Moreover, it is a homologue of the human Werner syndrome gene product WRN, a protein associated with premature ageing in humans.

SUMMARY DNA double-strand breaks (DSBs) activate a DNA damage response (DDR) that coordinates checkpoint pathways with DNA repair. ATM and ATR kinases are activated sequentially. Homology-directed repair (HDR) is initiated by resection of DSBs to generate 3′ ssDNA overhangs. How resection and HDR are activated during DDR or the roles of ATM and ATR in HDR are not known. Here, we show that CtIP undergoes ATR-dependent hyperphosphorylation in response to DSBs. ATR phosphorylates an invariant threonine, T818 of Xenopus CtIP (T859 in human). Non-phosphorylatable CtIP (T818A) does not bind to chromatin or initiate resection. Our data support a model in which ATM activity is required for an early step in resection leading to ATR activation, CtIP-T818 phosphorylation, and accumulation of CtIP on chromatin. Chromatin binding by modified CtIP precedes extensive resection and full checkpoint activation.