DNA replication is central to cell proliferation. Studies in the past six decades since the proposal of a semiconservative mode of DNA replication have confirmed the high degree of conservation of the basic machinery of DNA replication from prokaryotes to eukaryotes. However, the need for replication of a substantially longer segment of DNA in coordination with various internal and external signals in eukaryotic cells has led to more complex and versatile regulatory strategies. The replication program in higher eukaryotes is under a dynamic and plastic regulation within a single cell, or within the cell population, or during development. We review here various regulatory mechanisms that control the replication program in eukaryotes and discuss future directions in this dynamic field.

A novel approach to study electroweak physics at one-loop level in generic SU(2) L × U(1) Y theories is introduced. It separates the 1-loop corrections into two pieces: process specific ones from vertex and box contributions, and universal ones from contributions to the gauge boson propagators. The latter are parametrized in terms of four effective form factorsē 2 (q 2 ),s 2 (q 2 ),ḡ 2 Z (q 2 ) andḡ 2 W (q 2 ) corresponding to the γγ, γZ, ZZ and W W propagators. Under the assumption that only the Standard Model contributes to the process specific corrections, the magnitudes of the four form factors are determined at q 2 = 0 and at q 2 = m 2 Z by fitting to all available precision experiments. These values are then compared systematically with predictions of SU(2) L × U(1) Y theories. In all fits α s (m Z ) andᾱ(m 2 Z ) are treated as external parameters in order to keep the interpretation as flexible as possible. The treatment of the electroweak data is presented in detail together with the relevant theoretical formulae used to interpret the data. No deviation from the Standard Model has been identified. Ranges of the top quark and Higgs boson masses are derived as functions of α s (m Z ) andᾱ(m 2 Z ). Also discussed are consequences of the recent precision measurement of the left-right asymmetry at SLC as well as the impact of a top quark mass and an improved W mass measurement.To be published in Zeitschrift für Physik C into account by introducing appropriate external parameters. Up to this stage, our analysis is quite general, as the formulae are valid for any electroweak model respecting the flavor and chirality conservation laws of the SM, that is, for all new physics contributions which can interfere with the leading SM amplitudes.Although one may attempt to constrain these model-independent transition form factors directly by experiment, we find it impractical, since the number of independent 4 transition form factors exceeds by far the effective number of degrees of freedom provided by precision measurements. Hence, we perform the quantitative comparison of data with theory in a more restricted class of models which are minimal extensions from the SM, i.e. those models which respect the SU(2) L × U(1) Y gauge symmetry broken spontaneously down to U(1) EM .3: The transition form factors are expanded perturbatively in SU(2) L × U(1) Y gauge couplings, and radiative effects are classified either as the universal gauge boson propagator corrections or as the process specific vertex and box corrections. The universal propagator correction factors are then parameterized by four charge form factors,ē 2 (q 2 ),s 2 (q 2 ),ḡ 2 Z (q 2 ) andḡ 2 W (q 2 ), corresponding to the γγ, γZ, ZZ and W W propagator degrees of freedom.The restriction to the electroweak gauge group SU(2) L × U(1) Y implies at the tree level that all fermions, quarks and leptons, couple to the electroweak gauge bosons universally with the same coupling constant as long as they have common SU(2) L × U(1) Y quantum numbers. This universality of the g...

One of the long-standing questions in eukaryotic DNA replication is the mechanisms that determine where and when a particular segment of the genome is replicated. Cdc7/Hsk1 is a conserved kinase required for initiation of DNA replication and may affect the site selection and timing of origin firing. We identified rif1D, a null mutant of rif1 + , a conserved telomere-binding factor, as an efficient bypass mutant of fission yeast hsk1. Extensive deregulation of dormant origins over a wide range of the chromosomes occurs in rif1D in the presence or absence of hydroxyurea (HU). At the same time, many early-firing, efficient origins are suppressed or delayed in firing timing in rif1D. Rif1 binds not only to telomeres, but also to many specific locations on the arm segments that only partially overlap with the prereplicative complex assembly sites, although Rif1 tends to bind in the vicinity of the late/dormant origins activated in rif1D. The binding to the arm segments occurs through M to G1 phase in a manner independent of Taz1 and appears to be essential for the replication timing program during the normal cell cycle. Our data demonstrate that Rif1 is a critical determinant of the origin activation program on the fission yeast chromosomes.

Purpose: To investigate the diagnostic performance of circulating tumor cells (CTC) in discrimination between primary lung cancer and nonmalignant diseases as well as in prediction of distant metastasis. Patients and Methods: We prospectively evaluated CTCs in 7.5-mL samples of peripheral blood sampled from patients with a suspicion or a diagnosis of primary lung cancer. A semiautomated system was used to capture CTCs with an antibody against epithelial cell adhesion molecule. Results: Of 150 eligible patients, 25 were finally diagnosed as having nonmalignant disease, and 125 were diagnosed as having primary lung cancer with (n = 31) or without (n = 94) distant metastasis. CTCs were detected in 30.6% of lung cancer patients and in 12.0% of nonmalignant patients. CTC count was significantly higher in lung cancer patients than in nonmalignant patients, but a receiver operating characteristic (ROC) curve analysis showed an insufficient capability of the CTC test in discrimination between lung cancer and nonmalignant diseases with an area under ROC curve of 0.598 (95% confidence interval, 0.488-0.708; P = 0.122). Among lung cancer patients, CTC count significantly increased along with tumor progression, especially with development of distant metastasis. The area under ROC curve for CTC count in prediction of distant metastasis was 0.783 (95% confidence interval, 0.679-0.886; P < 0.001). When patients with one or more CTCs were judged as having metastatic disease, sensitivity and specificity of the CTC test were 71.0% and 83.0%, respectively. Conclusions: CTC is a useful surrogate marker of distant metastasis in primary lung cancer. (Clin Cancer Res 2009;15(22):6980-6)

Cdc7 kinase, conserved from yeasts to human, plays important roles in DNA replication. However, the mechanisms by which it stimulates initiation of DNA replication remain largely unclear. We have analyzed phosphorylation of MCM subunits during cell cycle by examining mobility shift on SDS-PAGE. MCM4 on the chromatin undergoes specific phosphorylation during S phase. Cdc7 phosphorylates MCM4 in the MCM complexes as well as the MCM4 N-terminal polypeptide. Experiments with phospho-amino acid-specific antibodies indicate that the S phase-specific mobility shift is due to the phosphorylation at specific N-terminal (S/T)(S/T)P residues of the MCM4 protein. These specific phosphorylation events are not observed in mouse ES cells deficient in Cdc7 or are reduced in the cells treated with siRNA specific to Cdc7, suggesting that they are mediated by Cdc7 kinase. The N-terminal phosphorylation of MCM4 stimulates association of Cdc45 with the chromatin, suggesting that it may be an important phosphorylation event by Cdc7 for activation of replication origins. Deletion of the N-terminal non-conserved 150 amino acids of MCM4 results in growth inhibition, and addition of amino acids carrying putative Cdc7 target sequences partially restores the growth. Furthermore, combination of MCM4 N-terminal deletion with alanine substitution and deletion of the N-terminal segments of MCM2 and MCM6, respectively, which contain clusters of serine/threonine and are also likely targets of Cdc7, led to an apparent nonviable phenotype. These results are consistent with the notion that the N-terminal phosphorylation of MCM2, MCM4, and MCM6 may play functionally redundant but essential roles in initiation of DNA replication.

Rif1 regulates replication timing and repair of double-strand DNA breaks. Using a chromatin immunoprecipitation-sequencing method, we identified 35 high-affinity Rif1-binding sites in fission yeast chromosomes. Binding sites tended to be located near dormant origins and to contain at least two copies of a conserved motif, CNWWGTGGGGG. Base substitution within these motifs resulted in complete loss of Rif1 binding and in activation of late-firing or dormant origins located up to 50 kb away. We show that Rif1-binding sites adopt G quadruplex-like structures in vitro, in a manner dependent on the conserved sequence and on other G tracts, and that purified Rif1 preferentially binds to this structure. These results suggest that Rif1 recognizes and binds G quadruplex-like structures at selected intergenic regions, thus generating local chromatin structures that may exert long-range suppressive effects on origin firing.

In the present study, we analyzed genomic alterations of BRCA1-associated protein 1 (BAP1) in 23 malignant mesotheliomas (MMs), 16 epithelioid and seven non-epithelioid, consisting of 18 clinical specimens and five established cell lines. In examining these samples for homozygous deletions and sequence-level mutations, we found biallelic BAP1 gene alterations in 14 of 23 MMs (61%). Seven of these 14 MMs had homozygous deletions of the partial or entire BAP1 gene, another five had sequencelevel mutations, including small deletions, a nonsense mutation, and missense mutations with additional monoallelic deletions, and the remaining two had homozygous mutations without allelic loss. All but one of the 14 BAP1 gene mutations were found in the epithelioid-type MMs; BAP1 mutations were found in 13 of 16 epithelioid-type MMs, but in only one of seven non-epithelioid-type MMs (13/16 vs 1/7; P = 0.005). There was no BAP1 mRNA expression in MMs with biallelic deletion and repressed expression was confirmed in MM specimens with deletion/mutation as compared with Met5a, SV40-transformed normal mesothelial cells. Western blot showed that seven of eight epithelioid MMs analyzed were BAP1 negative. Immunostaining with anti-BAP1 antibody in normal lung tissues revealed clear nuclear staining of normal mesothelial cells. No nuclear staining was observed among BAP1 mutation-positive MM tumors, whereas nuclear staining was observed among BAP1 mutation-negative MM tumors. These results suggest that the lack of the tumor suppressor BAP1 may be more specifically involved in the pathogenesis of epithelioid MM rather than non-epithelioid MM, and would be useful for diagnosis of epithelioid-type MM. (Cancer Sci 2012; 103: 868-874) M alignant mesothelioma (MM) is an asbestos-related malignancy that arises primarily from surface serosal cells of pleural, peritoneal, and pericardial cavities. Although the use of asbestos has decreased in Western countries and Japan, the incidence of MM is expected to increase over the next few decades because of the long latency period (20-40 years) of this malignancy.(1) Although the prognosis of MM is generally poor, epithelioid-type MM has been reported to be associated with better prognosis than non-epithelioid types of MM.(2) Multiple modality approaches involving surgery with radiation, chemotherapy, or immunotherapy have generated favorable outcomes, particularly for patients with epithelioid-type MM.(3)

We documented a significant increase in CTC count in drainage PV blood after surgical manipulation, especially in tumours with lymphatic invasion. We are awaiting survival data at 5 year follow-up examination, which may provide clinical significance of the pvCTC-count.